Methods and compositions in breast cancer diagnosis and therapeutics

ABSTRACT

The present invention is directed to compositions regarding a specific mutation in estrogen receptor alpha and their use as diagnostic markers in breast tissue, such as premalignant lesions, for the development of breast cancer. More specifically, cells of breast cancer whose nucleic acid comprises the estrogen receptor alpha mutation identify the breast cancer to be an invasive breast cancer.

This application claims priority to U.S. Ser. No. 60/304,018, filed Jul.9, 2001, U.S. Ser. No. 60/262,990, filed Jan. 19, 2001, and U.S. Ser.No. 10/052,092, filed Jan. 18, 2002, all of which are incorporatedherein in their entirety.

This invention was developed with funds from the United StatesGovernment. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention is directed to the fields of cancer and moleculargenetics. Specifically, the present invention is directed to thedetermination of susceptibility to breast cancer and the diagnosis ofinvasive breast cancer. More specifically, the present invention isdirected to a mutation in estrogen receptor alpha (ER) and itsassociation with breast cancer.

BACKGROUND OF THE INVENTION

Invasive breast cancer (IBC) is one of the most common and lethalmalignant neoplasms affecting women, especially in Western cultures. Themajority of IBCs are thought to develop over long periods of time fromcertain preexisting benign lesions. There are many types of benignlesions in the human breast, and only a few appear to have significantpremalignant potential. The most important premalignant lesionsrecognized today are referred to as a typical ductal hyperplasia (ADH),a typical lobular hyperplasia (ALH), ductal carcinoma in situ (DCIS),and lobular carcinoma in situ (LCIS). Although DCIS and LCIS possesssome malignant properties, such as loss of growth control, they lack theability to invade and metastasize and, in this sense, are premalignant.

A skilled artisan is aware that investigation of the role of theestrogen receptor in carcinomas is described by Watts et al., J. SteroidBiochem. Molec. Biol. 41(3), 529 (1992); Scott et al., J. Clinic.Invest. 88, 700 (1991); Ince et al., J. Bio. Chem. 268, 14026 (1993);Fuqua et al., Can. Res. 52, 43 (1992); McGuire et al., Mol. Endocr. 5,1571 (1991); Castles et al., Can. Res. 53, 5934 (1993); and Weigel anddeConinck, Can. Res. 53, 3472 (1993). Furthermore, description of theestrogen receptor mRNA may be found in Keaveney et al., J. Mol. Endocr.6, 111 (1991); Green et al., Nature 320, 134 (1986); White et al., Mol.Endocr. 1, 735 (1987); and Piva et al., J. Steroid Biochem. Molec. Biol.46, 531 (1993).

U.S. Pat. No. 6,162,606 is directed to identification of defectiveestrogen receptors associated with the classification of breast tumorswhich are responsive to or resistant to hormone therapy. Similarly, U.S.Pat. No. 5,563,035 regards monitoring the level of ERF-1, atranscriptional regulator of expression of the estrogen receptor, asbeing indicative of the response of a breast tumor to various therapies.

There is epidemiological evidence that there are genetic alterationsthat are closely associated with morphological tumor progression, suchas is found in studies in colon carcinoma (Vogelstein and Kinzler,1993). In this model (Dupont and Page, 1985), breast cancer ishypothesized as evolving from normal ductal epithelium to typicalhyperplasia, to a typical hyperplasia, to carcinoma in situ, to invasivecarcinoma, and finally to metastatic carcinoma. Recent data alsosuggests that the majority of hyperplasias share molecular alterationswith invasive disease in the same breast (O'Connell et al., 1998),providing genetic evidence that they are related. Unlike colon cancer,very little is known about the specific molecular changes that areassociated with the earliest stages of breast cancer evolution. However,it is likely that estrogens are important, since they are potentmitogens for normal breast epithelial cells, and it is believed that theduration of estrogen exposure to the breast epithelium is a significantrisk factor for breast cancer development. It is also generally agreedthat expression of the estrogen receptor (ER) is relatively low innormal breast epithelium, but is higher in certain premalignant lesions(e.g. typical hyperplasias) (van Agthoven et al., 1994).

Anandappa et al. (2000) detected no sequencing variants, such as singlebase change mutations, in ER from a panel of human primary breast cancerspecimens. However, Zhang et al. (1997) identified an ER mutant inmetastatic breast cancer which had a constitutive transactivationfunction independent of estradiol-binding.

Current human breast cancer management strategies utilize ER status as apredictive factor (McGuire, 1978; Burstein, 1982; Brooks et al., 1980;Degenshein et al., 1980; McGuire et al., 1975; McGuire, 1987; Elledgeand McGuire, 1993; Gelbfish et al., 1988; Williams et al., 1987; Kohailet al., 1985; Donegan, 1992; Millis, 1980; McCarty et al., 1980),although none regard the specific mutation of the present invention.Present human breast tumor tissue specimens are subjected to bothligand-binding studies and immunohistochemical analyses to determine ERstatus (King et al., 1979; Shousha et al., 1989; Shousha et al., 1990).Thus, as has been acknowledged (see, for example, Roger et al., 2000),the art presently lacks a molecular marker for breast tissue, such as apremalignant lesion, which is at risk for breast cancer, particularlyfor invasive breast cancer, and also lacks a marker for the purpose ofimproving approaches to risk prediction and treatment strategies.Identification of a specific molecular marker for an altered ER as anearly event in breast cancer evolution would be a significant advance inthe field and would provide an ideal diagnosis tool for the detection ofsusceptibility to breast cancer and its subsequent prevention.

SUMMARY OF THE INVENTION

In an embodiment of the present invention there is an isolated estrogenreceptor alpha nucleic acid sequence comprising an A908G mutation.

In another embodiment of the present invention there is an isolatedestrogen receptor alpha amino acid sequence comprising a K303Rsubstitution.

In an additional embodiment of the present invention there is a methodof detecting susceptibility to development of breast cancer in anindividual, comprising the steps of obtaining a sample from a breast ofthe individual, wherein the sample comprises a cell having an estrogenreceptor alpha nucleic acid sequence; and assaying the nucleic acidsequence for an A908G mutation, wherein the presence of the mutation inthe nucleic acid sequence indicates the individual has breast cancer. Ina specific embodiment, the sample is from a premalignant lesion of thebreast.

In an additional embodiment of the present invention there is a methodof detecting susceptibility to development of invasive breast cancer inan individual, comprising the steps of obtaining a sample from a breastof the individual; and assaying an estrogen receptor alpha nucleic acidsequence from a cell of the sample for an A908G mutation, wherein thepresence of the mutation in the nucleic acid sequence detectssusceptibility of the premalignant lesion to develop into the invasivebreast cancer. In a specific embodiment, the sample is from apremalignant lesion of the breast.

In an additional embodiment of the present invention there is a methodof detecting susceptibility to development of invasive breast cancerfrom a premalignant lesion in a breast, comprising the steps ofobtaining a sample from the premalignant lesion; dissecting the sampleto differentiate hyperplastic cells in the sample from nonhyperplasticcells; and assaying an estrogen receptor alpha nucleic acid sequencefrom the hyperplastic cell of the sample for an A908G mutation, whereinthe presence of the mutation in the nucleic acid sequence detectssusceptibility of the premalignant lesion to develop into the invasivebreast cancer. In a specific embodiment, the dissection step comprisesremoval of the hyperplastic cells from the sample by manual manipulationor by laser capture microdissection. In another specific embodiment, thesample is obtained by biopsy. In a specific embodiment, the assayingstep comprises sequencing, single stranded conformation polymorphism,mismatch oligonucleotide mutation detection, or a combination thereof.In an additional specific embodiment, the assaying step is by antibodydetection with antibodies to the A908G mutation of the estrogen receptoralpha nucleic acid sequence or is by antibody detection with antibodiesto an acetylated estrogen receptor alpha amino acid sequence.

In an additional embodiment of the present invention there is a methodof classifying breast cancer in an individual, comprising the steps ofobtaining from the individual a sample from the breast, wherein thesample contains a cancer cell; and assaying an estrogen receptor alphanucleic acid sequence from the cell of the sample for an A908G mutation,wherein the presence of the mutation identifies the breast cancer to beinvasive breast cancer. In a specific embodiment, the sample is obtainedby biopsy. In another specific embodiment, the assaying step is selectedfrom the group consisting of sequencing, single stranded conformationpolymorphism, mismatch oligonucleotide mutation detection, and acombination thereof. In an additional specific embodiment the assayingstep is by antibody detection with antibodies to the A908G mutation ofthe estrogen receptor alpha nucleic acid sequence or by antibodydetection with antibodies to an acetylated estrogen receptor alpha aminoacid sequence.

In another embodiment of the present invention there is a method ofdiagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual, wherein the samplecomprises a cell having an estrogen receptor alpha nucleic acidsequence; and assaying the nucleic acid sequence for an A908G mutation,wherein the presence of the mutation in the nucleic acid sequenceindicates the individual has breast cancer.

In another embodiment of the present invention there is a method ofdiagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; dissecting thesample to differentiate a cell suspected of being cancerous from anoncancerous cell; and assaying the cell suspected of being cancerousfor an A908G mutation in an estrogen receptor alpha nucleic acidsequence, wherein the presence of the mutation in the nucleic acidsequence indicates the individual has breast cancer. In a specificembodiment, the dissection step comprises removal of the cells suspectedof being cancerous from the sample by manual manipulation or by lasercapture microdissection. In a specific embodiment, the sample isobtained by biopsy. In another specific embodiment, the assaying step isselected from the group consisting of sequencing, single strandedconformation polymorphism, mismatch oligonucleotide mutation detection,and a combination thereof. In an additional specific embodiment, theassaying step is by antibody detection with antibodies to the A908Gmutation of the estrogen receptor alpha nucleic acid sequence or is byantibody detection with antibodies to an acetylated estrogen receptoralpha amino acid sequence.

In another embodiment of the present invention there is a kit fordiagnosing an A908G mutation in an estrogen receptor alpha nucleic acidsequence, comprising at least one primer selected from the groupconsisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In one embodiment, theprimers are extendable. In an alternative embodiment, the primers arenonextendable.

In another embodiment of the present invention there is a monoclonalantibody that binds immunologically to an acetylated estrogen receptoralpha amino acid sequence, or an antigenic fragment thereof.

In another embodiment of the present invention there is a monoclonalantibody that binds immunologically to an A908G mutation in an estrogenreceptor alpha nucleic acid sequence.

In an additional embodiment of the present invention there is a methodto correct a G mutation at nucleotide 908 of an estrogen receptor alphanucleic acid sequence in a cell of an individual, comprising the step ofadministering to the cell an estrogen receptor alpha nucleic acidsequence comprising an A at nucleotide 908. In a specific embodiment,the estrogen receptor alpha nucleic acid sequence comprising an A atnucleotide 908 is present on a vector. In another specific embodiment,the vector is selected from the group consisting of plasmid, viralvector, liposome, and a combination thereof. In an additional specificembodiment, the viral vector is selected from the group consisting ofadenoviral vector, retroviral vector, adeno-associated viral vector, ora combination thereof.

In an additional embodiment of the present invention there is a methodto prevent breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; identifying in thesample an A908G mutation in a nucleic acid sequence of estrogen receptoralpha; and correcting the A908G mutation, wherein the correction resultsin the prevention of the breast cancer. In a specific embodiment, thebreast sample is from a premalignant lesion of the breast. In anotherspecific embodiment, the correction step comprises administering anestrogen receptor alpha nucleic acid sequence comprising a G atnucleotide 908 to a cell comprising an estrogen receptor alpha nucleicacid sequence containing the A908G mutation.

In an additional embodiment of the present invention there is a methodto treat breast cancer in an individual, wherein an estrogen receptoralpha nucleic acid sequence in a breast cell of the individual has anA908G mutation, comprising the step of administering to the cell anestrogen receptor alpha nucleic acid sequence comprising a G atnucleotide 908.

In another embodiment of the present invention there is a method toprevent breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; identifying in thesample an arginine at amino acid residue 303 in an amino acid sequenceof estrogen receptor alpha; and administering to the individual an aminoacid sequence of estrogen receptor alpha comprising a lysine at aminoacid residue 303, wherein the administration results in the preventionof the breast cancer. In a specific embodiment, the breast sample isfrom a premalignant lesion of the breast.

In an object of the present invention there is a method of identifying amodulator of an estrogen receptor alpha K303R polypeptide, comprisingproviding a candidate modulator; admixing the candidate modulator withan isolated compound or cell, or a suitable experimental animal;measuring one or more characteristics of the compound, cell or animal;and comparing the characteristic measured with the characteristic of thecompound, cell or animal in the absence of the candidate modulator,wherein a difference between the measured characteristics indicates thatthe candidate modulator is the modulator of the compound, cell oranimal.

In another object of the present invention, there is a method ofscreening for a modulator of an estrogen receptor alpha polypeptidecomprising a K303R substitution, comprising introducing to a cell avector comprising a nucleic acid sequence which encodes the estrogenreceptor alpha K303R polypeptide; a vector comprising at least oneestrogen-responsive regulatory element operatively linked to a reporterpolynucleotide; and a test agent; and assaying expression of thereporter polynucleotide in the presence of the test agent, wherein thetest agent is the modulator when the reporter polynucleotide expressionchanges in the presence of the test agent. In a specific embodiment, atleast one of the vectors is transiently transfected into the cell. Inanother specific embodiment, at least one of the vectors is stablytransfected into the cell. In an additional embodiment, when expressionof the reporter polynucleotide is upregulated, the modulator is anagonist. In an additional embodiment, when expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In afurther specific embodiment, when the expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In aspecific embodiment, the cell is a mammalian cell. In a further specificembodiment, the mammalian cell is selected from the group consisting ofCHO, HepG2, HeLa, COS-1, MCF-7, MDA-MB-231, T47D, ZR-75, MDA-MB-435,BT-20, MDA-MB-468, and HEC-1. In an additional specific embodiment, theestrogen-responsive regulatory element is selected from the groupconsisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49;SEQ ID NO:22; SEQ ID NO:26, and SEQ ID NO:8. In an additional specificembodiment, the reporter polynucleotide is luciferase, chloramphenicolacetyltransferase, renilla or β-galactosidase. In a specific embodiment,there is a method of treating breast cancer in an individual comprisingthe step of administering the antagonist to the individual.

In another object of the present invention, there is a method ofidentifying a polypeptide which interacts with an estrogen receptoralpha polypeptide comprising a K303R substitution, comprisingintroducing to a cell, a vector comprising a polynucleotide whichencodes a chimeric polypeptide comprising the estrogen receptor alphaK303R polypeptide and a DNA binding domain; introducing to the cell, avector comprising a polynucleotide which encodes a chimeric polypeptidecomprising a candidate polypeptide and a DNA activation domain; andassaying for an interaction between the DNA binding domain and the DNAactivation domain, wherein when the interaction occurs, the candidatepolypeptide is the polypeptide which interacts with the estrogenreceptor alpha K303R polypeptide. In a specific embodiment, thepolypeptide which interacts with the estrogen receptor alpha K303Rpolypeptide is an antagonist of the estrogen receptor alpha K303Rpolypeptide. In a specific embodiment, the interaction is assayed byassaying for a change in expression of a reporter sequence. In aspecific embodiment, the cell is a yeast cell. In another specificembodiment, the cell is a mammalian cell. In a further specificembodiment, the DNA activation domain and the DNA binding domain arefrom GAL4 or LexA. In an additional specific embodiment, the reportersequence is selected from the group consisting of β-galactosidase,luciferase, chloramphenicol acetyltransferase, and renilla. In aspecific embodiment, there is a method of treating an individual forbreast cancer, comprising administering the antagonist to theindividual.

In another object of the present invention, there is a method ofidentifying a peptide which interacts with an estrogen receptor alphaK303R polypeptide, comprising obtaining an estrogen receptor alpha K303Rpolypeptide having an affinity tag and a label; introducing thepolypeptide to a substrate comprising a plurality of bacteriophage,wherein the bacteriophage produce a candidate peptide; and determiningbinding of the polypeptide with the candidate peptide, wherein when thepolypeptide binds the candidate peptide, the candidate peptide is theinteracting peptide. In a specific embodiment, the label is a colorlabel, a fluorescence label, or a radioactive label. In another specificembodiment, the affinity tag is biotin, GST, histidine, myc, orcalmodulin-binding protein.

In an additional object of the present invention, there is a method ofidentifying a compound for the treatment of breast cancer associatedwith an estrogen receptor alpha K303R polypeptide, comprising the stepsof obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity. In a specificembodiment, the compound having the activity is an antagonist of theestrogen receptor alpha K303R polypeptide. In a specific embodiment, themethod further comprises dispersing the compound in a pharmaceuticalcarrier; and administering a therapeutically effective amount of thecompound in the carrier to an individual having the breast cancer.

Another object of the present invention is the compound obtained by themethod of identifying a compound for the treatment of breast cancerassociated with an estrogen receptor alpha K303R polypeptide, comprisingthe steps of obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity.

An additional object of the present invention is a pharmacologicallyacceptable composition comprising the compound obtained by the method ofidentifying a compound for the treatment of breast cancer associatedwith an estrogen receptor alpha K303R polypeptide, comprising the stepsof obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity; and a pharmaceuticalcarrier.

Other and further objects, features, and advantages would be apparentand eventually more readily understood by reading the followingspecification and be reference to the accompanying drawings forming apart thereof, or any examples of the presently preferred embodiments ofthe invention given for the purpose of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates examples of typical estrogen receptor (ER) expressionin premalignant breast lesions as assayed by immunohistochemistry (smalldark nuclei are ER-positive cells).

FIG. 2 illustrates sequence analysis of ER Variant (VAR) and Wild-Type(WT) cDNAs isolated from frozen breast hyperplastic tissue. A portion ofthe sequencing products are shown for wild-type and variant clonesdemarcating the location of the G transition and Arg substitution. ERdomains A through E and the exons across these domains are shown on thebottom panel with the location of the Lys to Arg change demarcated witha box across exon 4 at the end of domain D.

FIG. 3 demonstrates detection of the ER VAR sequence in archival breastspecimens by identification of WT and VAR ER sequences in one patientwith typical hyperplasia (TH). Normal adjacent breast epithelium (NAdj.), TH, and distant normal epithelium (N Dis.) were all available foranalysis from this patient. The position of the A908G sequence isindicated by arrows.

FIG. 4 illustrates growth curves of stable MCF-7 transfectants inresponse to increasing concentrations of estradiol in the media. Cellswere plated at a density of 2×10⁴ in media containing 10%charcoal-stripped, estrogen-free fetal calf serum and were either leftuntreated [▪] or treated with the indicated estradiol concentrations(1×10⁻¹²[●], 1×10⁻¹¹ [π], 1×10⁻⁹[♦] M). The medium was replaced every 48h and the cells were harvested and counted on days 2, 4, 6, and 8,respectively. Cell number×10⁴ is shown. Panel A demonstratesuntransfected parental MCF-7 cells. Panel B demonstrates vector-alonestably transfected cells. Panels C and D demonstrate cells stablytransfected with WT ER. Panels E, F, and D demonstrate cells stablytransfected with the mutant ER.

FIG. 5 demonstrates interaction of the WT and mutant ERs with SRC-1,SRC-2 and SRC-3 in vitro.

FIG. 6 demonstrates detection of the ER Mutant (Mut) in archival breastspecimens, including identification of WT and Mut ER alleles in 10typical breast hyperplasias. Both Mut and WT plasmid DNAs were includedas positive controls for the location of the migration of theirrespective alleles (first two lanes). The ten hyperplastic lesions arelabeled 1 through 10.

FIG. 7 illustrates oligonucleotide mismatch hybridization of one patientwith concurrent breast lesions. Laser capture microdissection was usedto precisely microdissect with an enrichment of >90% cellularity.PCR-amplified fragments were obtained from normal breast epitheliumadjacent to a hyperplasia (AB), normal breast epithelium distant frommalignant breast lesions (DB), TH, normal skin (NS) and two differentDCIS lesions (DCIS 1 and 2) and slotted in duplicate onto nylonmembranes (Micro Separation, Inc., Westboro, Mass.). The panel on theleft was hybridized with an oligonucleotide to the WT ER sequence, whilethe panel on the right was hybridized with an oligonucleotide specificfor the Mut sequence.

FIGS. 8A through 8D demonstrates ductal hyperplasias in K303R transgenicmice. FIGS. 8E-8F show nontransgenic mammary gland controls.

FIG. 9 shows a comparison of ductal epithelium from K303R transgenicmice versus nontransgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

It will be readily apparent to one skilled in the art that variousembodiments and modifications may be made in the invention disclosedherein without departing from the scope and spirit of the invention.

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

I. Definitions

The term “A908G mutation” as used herein is defined as an adenine(A)-to-guanine (G) base pair transition at nucleotide position 908 in anestrogen receptor alpha nucleic acid sequence, relative to the firstnucleotide of the first codon of the translated amino acid sequence. Askilled artisan recognizes that multiple estrogen receptor alpha nucleicacid sequences exist which are, for example, alternative splicevariants. Thus, there are some estrogen receptor alpha nucleic acidsequences of different sizes, and the A908G mutation which is present atnucleotide (nt) 908 in the full-length mutated sequence may no longer beat position 908 in a variant sequence. However, a skilled artisan canreadily identify the equivalent or analogous sequence in these variantsby sequence homology and comparison, and/or by analyzing locations,arrangements or relationships of splicing manipulations. Thus, anestrogen receptor alpha nucleic acid sequence which contains theindicated mutation yet is a variant, such as an alternatively splicedform of the sequence, is still within the scope of the presentinvention.

The term “agonist” as used herein is defined as a compound orcomposition which promotes, facilitates, allows, induces, or otherwiseassists, activates or increases the function of the estrogen receptoralpha K303R polypeptide.

The term “antagonist” as used herein is defined as a compound orcomposition which inhibits, stops, deters, impedes, delays, or otherwiseprevents the activity and functioning of the estrogen receptor alphaK303R polypeptide.

The term “biopsy” as used herein is defined as removal of a tissue froma breast for the purpose of examination, such as to establish diagnosis.Examples of types of biopsies include by application of suction, such asthrough a needle attached to a syringe; by instrumental removal of afragment of tissue; by removal with appropriate instruments through anendoscope; by surgical excision, such as of the whole lesion; and thelike.

The term “breast cancer” as used herein is defined as cancer whichoriginates in the breast. In a specific embodiment, the breast cancerspreads to other organs, such as lymph nodes. In a specific embodiment,the breast cancer is invasive and may be metastatic.

The term “cancer” as used herein is defined as a new growth of tissuecomprising uncontrolled and progressive multiplication. In a specificembodiment, upon a natural course the cancer is fatal. In specificembodiments, the cancer is invasive, metastatic, and/or anaplastic (lossof differentiation and of orientation to one another and to their axialframework).

The term “invasive” as used herein refers to cells which have theability to infiltrate surrounding tissue. In a specific embodiment, theinfiltration results in destruction of the surrounding tissue. Inanother specific embodiment, the cells are cancer cells. In a preferredembodiment, the cells are breast cancer cells, and the cancer spreadsout of a duct into surrounding breast epithelium. In a specificembodiment, “metastatic” breast cancer is within the scope of“invasive.”

The term “K303R substitution” as used herein is defined as the aminoacid substitution which results from the A908G mutation in estrogenreceptor alpha nucleic acid sequence. The term “Lys303Arg substitution”is used herein interchangeably. A skilled artisan recognizes thatmultiple estrogen receptor alpha amino acid sequences exist which are,for example, alternative splice variants. Thus, there are some estrogenreceptor alpha amino acid sequences of different sizes, and the K303Rsubstitution which is present in the full-length mutated sequence may nolonger be at position 303 in the variant sequence. However, a skilledartisan can readily identify the equivalent or analogous sequence inthese variants by sequence homology and comparison, and/or by analyzinglocations, arrangements or relationships of splicing manipulations.Thus, an estrogen receptor alpha amino acid sequence which contains theindicated mutation yet is a variant, such as an alternatively splicedform of the sequence, is still within the scope of the presentinvention.

The term “laser capture microdissection” as used herein is defined asthe use of an infrared (IR) laser beam to remove a desired cell from anondesired cell. In preferred embodiments, the desired cell is a cancercell and the nondesired cell is a normal cell. In another preferredembodiment, the cancer cell is a breast cancer cell.

The term “manual manipulation” as used herein is defined as theselective removal of a desired cell or cells from a nondesired cell orcells by hand. In preferred embodiments, the desired cell is a cancercell and the nondesired cell is a normal cell. In another preferredembodiment, the cancer cell is a breast cancer cell.

The term “metastatic” as used herein is defined as the transfer ofcancer cells from one organ or part to another not directly connectedwith it. In a specific embodiment, breast cancer cells spread to anotherorgan or body part, such as lymph nodes.

The term “premalignant lesion” as used herein is defined as a collectionof cells in a breast with histopathological characteristics whichsuggest at least one of the cells has an increased risk of becomingbreast cancer. A skilled artisan recognizes that the most importantpremalignant lesions recognized today include unfolded lobules (UL;other names: blunt duct adenosis, columnar alteration of lobules), usualductal hyperplasia (UDH; other names: proliferative disease withoutatypia, epitheliosis, papillomatosis, benign proliferative disease), atypical ductal hyperplasia (ADH), a typical lobular hyperplasia (ALH),ductal carcinoma in situ (DCIS), and lobular carcinoma in situ (LCIS).Other lesions which may have premalignant potential include intraductalpapillomas, sclerosisng adenosis, and fibroadenomas (especially atypical fibroadenomas). In a specific embodiment, the collection ofcells is a lump, tumor, mass, bump, bulge, swelling, and the like. Otherterms in the art which are interchangeable with “premalignant lesion”include premalignant hyperplasia, premalignant neoplasia, and the like.

The term “sample from a breast” as used herein is defined as a specimenfrom any part or tissue of a breast. A skilled artisan recognizes thatthe sample may be obtained by any method, such as biopsy. In a specificembodiment the sample is obtained by nipple aspirate (see, for example,Sauter et al. (1997)). In another specific embodiment, the sample isfrom hyperplastic or malignant breast epithelium. In a specificembodiment, the sample is from the epithelium. In another specificembodiment, the sample is from a premalignant lesion. A skilled artisanrecognizes that within the scope of the present invention is theembodiment wherein a normal, or benign, sample, such as from anepithelium, is obtained for risk screening.

II. The Present Invention

The best current model of breast cancer evolution suggests that mostcancers arise from certain premalignant lesions. The present inventionis directed to a common (34%) somatic mutation in the estrogen receptor(ER) α gene in a series of 59 typical hyperplasias, a type of earlypremalignant breast lesion. The mutation, which affects the border ofthe hinge and hormone binding domains of ERα, showed increasedsensitivity to estrogen as compared to wild-type ERα in stablytransfected breast cancer cells, including markedly increasedproliferation at subphysiologic levels of estrogen. The mutated ERαexhibits significantly enhanced binding to the TIF-2 (SRC-2) and SRC-3co-activators and moderately enhanced binding to SRC-1 at low levels ofhormone, which in a specific embodiment explains its increased estrogenresponsiveness. In a preferred embodiment, this mutation promotes oraccelerates the development of cancer from premalignant breast lesions.As such, it is a useful tool for the diagnosis of breast cancer anddetermination of susceptibility to the development of breast cancer,including determination of the propensity for invasiveness.

A skilled artisan recognizes the existence of a variety of inherited, orsomatically acquired, variations in the DNA of the estrogen receptoralpha gene in cells in a breast sample, which, in the latter case, maydiffer in a mixture of normal and neoplastic cells. As demonstrated inthe Examples herein, those cells having DNA that contain an A908Gmutation in the estrogen receptor alpha nucleic acid sequence are orwill become cancerous, and particularly will be a cell of a breastcancer which will become metastatic. The present invention is directedto methods and compositions related to detection of the A908G mutation.

In an embodiment of the present invention there is an isolated estrogenreceptor alpha nucleic acid sequence comprising an A908G mutation.

In another embodiment of the present invention there is an isolatedestrogen receptor alpha amino acid sequence comprising a K303Rsubstitution.

In an additional embodiment of the present invention there is a methodof detecting susceptibility to development of breast cancer in anindividual, comprising the steps of obtaining a sample from a breast ofthe individual, wherein the sample comprises a cell having an estrogenreceptor alpha nucleic acid sequence; and assaying the nucleic acidsequence for an A908G mutation, wherein the presence of the mutation inthe nucleic acid sequence indicates the individual has breast cancer. Ina specific embodiment, the sample is from a premalignant lesion of thebreast.

In an additional embodiment of the present invention there is a methodof detecting susceptibility to development of invasive breast cancer inan individual, comprising the steps of obtaining a sample from a breastof the individual; and assaying an estrogen receptor alpha nucleic acidsequence from a cell of the sample for an A908G mutation, wherein thepresence of the mutation in the nucleic acid sequence detectssusceptibility of the premalignant lesion to develop into the invasivebreast cancer. In a specific embodiment, the sample is from apremalignant lesion of the breast.

In an additional embodiment of the present invention there is a methodof detecting susceptibility to development of invasive breast cancerfrom a premalignant lesion in a breast, comprising the steps ofobtaining a sample from the premalignant lesion; dissecting the sampleto differentiate hyperplastic cells in the sample from nonhyperplasticcells; and assaying an estrogen receptor alpha nucleic acid sequencefrom the hyperplastic cell of the sample for an A908G mutation, whereinthe presence of the mutation in the nucleic acid sequence detectssusceptibility of the premalignant lesion to develop into the invasivebreast cancer. In a specific embodiment, the dissection step comprisesremoval of the hyperplastic cells from the sample by manual manipulationor by laser capture microdissection. In another specific embodiment, thesample is obtained by biopsy. In a specific embodiment, the assayingstep comprises sequencing, single stranded conformation polymorphism,mismatch oligonucleotide mutation detection, or a combination thereof.In an additional specific embodiment, the assaying step is by antibodydetection with antibodies to the A908G mutation of the estrogen receptoralpha nucleic acid sequence or is by antibody detection with antibodiesto an acetylated estrogen receptor alpha amino acid sequence. In afurther specific embodiment, the assaying step is by detection of SNPsby methods well known in the art.

In an additional embodiment of the present invention there is a methodof classifying breast cancer in an individual, comprising the steps ofobtaining from the individual a sample from the breast, wherein thesample contains a cancer cell; and assaying an estrogen receptor alphanucleic acid sequence from the cell of the sample for an A908G mutation,wherein the presence of the mutation identifies the breast cancer to beinvasive breast cancer. In a specific embodiment, the sample is obtainedby biopsy. In another specific embodiment, the assaying step is selectedfrom the group consisting of sequencing, single stranded conformationpolymorphism, mismatch oligonucleotide mutation detection, and acombination thereof.

A skilled artisan recognizes that there are a variety of methods todetect a mutation in a nucleic acid sequence in addition to thesemethods. Methods regarding allele-specific probes for analyzingparticular nucleotide sequences are described by e.g., Saiki et al.,Nature 324, 163-166 (1986); Dattagupta, EP 235,726 (U.S. Pat. No.836,378 (Mar. 5, 1986); U.S. Pat. No. 943,006 (Dec. 29, 1986)); Saiki,WO 89/11548 (U.S. Pat. No. 197,000 (May 20, 1988); U.S. Pat. No. 347,495(May 4, 1989)). Allele-specific probes are typically used in pairs. Onemember of the pair shows perfect complementarity to a wildtype alleleand the other members to a variant allele. In idealized hybridizationconditions to a homozygous target, such a pair shows an essentiallybinary response. That is, one member of the pair hybridizes and theother does not. An allele-specific primer hybridizes to a site on targetDNA overlapping the particular site in question and primes amplificationof an allelic form to which the primer exhibits perfect complementarily(Gibbs, 1989). This primer is used in conjunction with a second primerwhich hybridizes at a distal site. Amplification proceeds from the twoprimers leading to a detectable product signifying the particularallelic form is present. A control is usually performed with a secondpair of primers, one of which shows a single base mismatch at thepolymorphic site and the other of which exhibits perfect complementarilyto a distal site. The single-base mismatch impairs amplification andlittle, if any, amplification product is generated.

Particular nucleic acid sites can also be identified by hybridization tooligonucleotide arrays. An example is described in WO 95/11995, whichincludes arrays having four probe sets. A first probe set includesoverlapping probes spanning a region of interest in a referencesequence. Each probe in the first probe set has an interrogationposition that corresponds to a nucleotide in the reference sequence.That is, the interrogation position is aligned with the correspondingnucleotide in the reference sequence when the probe and referencesequence are aligned to maximize complementarily between the two. Foreach probe in the first set, there are three corresponding probes fromthree additional probe sets. Thus, there are four probes correspondingto each nucleotide in the reference sequence. The probes from the threeadditional probe sets are identical to the corresponding probe from thefirst probe set except at the interrogation position, which occurs inthe same position in each of the four corresponding probes from the fourprobe sets, and is occupied by a different nucleotide in the four probesets. Such an array is hybridized to a labeled target sequence, whichmay be the same as the reference sequence, or a variant thereof. Theidentity of any nucleotide of interest in the target sequence can bedetermined by comparing the hybridization intensities of the four probeshaving interrogation positions aligned with that nucleotide. Thenucleotide in the target sequence is the complement of the nucleotideoccupying the interrogation position of the probe with the highesthybridization intensity.

WO 95/11995 also describes subarrays that are optimized for detection ofvariant forms of a precharacterized nucleotide site. A subarray containsprobes designed to be complementary to a second reference sequence,which can be an allelic variant of the first reference sequence. Thesecond group of probes is designed by the same principles as aboveexcept that the probes exhibit complementarity to the second referencesequence. The inclusion of a second group can be particularly useful foranalyzing short subsequences of the primary reference sequence in whichmultiple mutations are expected to occur within a short distancecommensurate with the length of the probes (i.e., two or more mutationswithin 9 to 21 bases).

An additional strategy for detecting a particular nucleotide site usesan array of probes is described in EP 717,113 (U.S. Pat. No. 327,525(Oct. 21, 1994). In this strategy, an array contains overlapping probesspanning a region of interest in a reference sequence. The array ishybridized to a labeled target sequence, which may be the same as thereference sequence or a variant thereof. If the target sequence is avariant of the reference sequence, probes overlapping the site ofvariation show reduced hybridization intensity relative to other probesin the array. In arrays in which the probes are arranged in an orderedfashion stepping through the reference sequence (e.g., each successiveprobe has one fewer 5′ base and one more 3′ base than its predecessor),the loss of hybridization intensity is manifested as a “footprint” ofprobes approximately centered about the point of variation between thetarget sequence and reference sequence.

Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses a methodfor determining the identity of the nucleotide present at a particularsite that employs a specialized exonuclease-resistant nucleotidederivative. A primer complementary to the allelic sequence immediately3′ to the site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the site on the target moleculecontains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the site of the target molecule was complementaryto that of the nucleotide derivative used in the reaction. The Mundymethod has the advantage that it does not require the determination oflarge amounts of extraneous sequence data. It has the disadvantages ofdestroying the amplified target sequences, and unmodified primer and ofbeing extremely sensitive to the rate of polymerase incorporation of thespecific exonuclease-resistant nucleotide being used.

Cohen, D. et al. (French Patent 2,650,840 (U.S. Pat. No. 4,420,902 (Dec.20, 1983)); PCT Appln. No. WO91/02087) discuss a solution-based methodfor determining the identity of the nucleotide of a particular site. Asin the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to the site.The method determines the identity of the nucleotide of that site usinglabeled dideoxynucleotide derivatives, which, if complementary to thenucleotide of the site will become incorporated onto the terminus of theprimer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712 (U.S. Pat. No.664,837 (Mar. 5, 1991); U.S. Pat. No. 775,786 (Oct. 11, 1991)). Themethod of Goelet, P. et al. uses mixtures of labeled terminators and aprimer that is complementary to the sequence 3′ to a site in question.The labeled terminator that is incorporated is thus determined by, andcomplementary to, the nucleotide present in the site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase. It isthus easier to perform, and more accurate than the method discussed byCohen.

An alternative approach, the “Oligonucleotide Ligation Assay” (“OLA”)(Landegren, U. et al., Science 241:1077-1080 (1988)) has also beendescribed as capable of detecting a nucleotide sequence variation. TheOLA protocol uses two oligonucleotides which are designed to be capableof hybridizing to abutting sequences of a single strand of a target. Oneof the oligonucleotides is biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA. In addition to requiring multiple, andseparate, processing steps, one problem associated with suchcombinations is that they inherit all of the problems associated withPCR and OLA.

Recently, several primer-guided nucleotide incorporation procedures forassaying particular sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syv anen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)).

In an additional specific embodiment of the present invention anassaying step is by antibody detection with antibodies to the A908Gmutation of the estrogen receptor alpha nucleic acid sequence or byantibody detection with antibodies to an acetylated estrogen receptoralpha amino acid sequence.

In another embodiment of the present invention there is a method ofdiagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual, wherein the samplecomprises a cell having an estrogen receptor alpha nucleic acidsequence; and assaying the nucleic acid sequence for an A908G mutation,wherein the presence of the mutation in the nucleic acid sequenceindicates the individual has breast cancer.

In another embodiment of the present invention there is a method ofdiagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; dissecting thesample to differentiate a cell suspected of being cancerous from anoncancerous cell; and assaying the cell suspected of being cancerousfor an A908G mutation in an estrogen receptor alpha nucleic acidsequence, wherein the presence of the mutation in the nucleic acidsequence indicates the individual has breast cancer. In a specificembodiment, the dissection step comprises removal of the cells suspectedof being cancerous from the sample by manual manipulation or by lasercapture microdissection. In a specific embodiment, the sample isobtained by biopsy. In another specific embodiment, the assaying step isselected from the group consisting of sequencing, single strandedconformation polymorphism, mismatch oligonucleotide mutation detection,and a combination thereof. In an additional specific embodiment, theassaying step is by antibody detection with antibodies to the A908Gmutation of the estrogen receptor alpha nucleic acid sequence or is byantibody detection with antibodies to an acetylated estrogen receptoralpha amino acid sequence. In a specific embodiment, the mutation isdetected by SNP analysis, using standard methods in the art. Somemethods use extendable primers for incorporating radiolabelednucleotides, which can then be detected by fluorescence or resonance.For example, PerkinElmer™ (Shelton, Conn.) has the AcycloPrime™fluorescence polarization SNP detection system which utilizes terminatorlabeled nucleotides to facilitate detection of the SNP upon fluorescencepolarization. Also, Applied Biosystems (Foster City, Calif.) has the ABIPRISM® turbo TaqMan® probes for genotyping by allelic detection whichutilizes fluorescent dyes, such as VIC™, and TET and 6-FAM, fordetection. In a specific embodiment, the thymidine residues of theprobes are replaced with 5-propyne-2′-deoxyuridine, which increases theT_(m) of these probes by approximately 1° C. per substitution andfacilitates design of a shorter probe for greater accuracy.

In another embodiment of the present invention there is a kit fordiagnosing an A908G mutation in an estrogen receptor alpha nucleic acidsequence, comprising at least one primer selected from the groupconsisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In a specific embodiment,the kit contains primers which are extendable. In an alternativespecific embodiment, the kit contains primers which are nonextendable.

In another embodiment of the present invention there is a monoclonalantibody that binds immunologically to an acetylated estrogen receptoralpha amino acid sequence, or an antigenic fragment thereof

In another embodiment of the present invention there is a monoclonalantibody that binds immunologically to an A908G mutation in an estrogenreceptor alpha nucleic acid sequence.

In an additional embodiment of the present invention there is a methodto correct a G mutation at nucleotide 908 of an estrogen receptor alphanucleic acid sequence in a cell of an individual, comprising the step ofadministering to the cell an estrogen receptor alpha nucleic acidsequence comprising an A at nucleotide 908. In a specific embodiment,the estrogen receptor alpha nucleic acid sequence comprising an A atnucleotide 908 is present on a vector. In another specific embodiment,the vector is selected from the group consisting of plasmid, viralvector, liposome, and a combination thereof. In an additional specificembodiment, the viral vector is selected from the group consisting ofadenoviral vector, retroviral vector, adeno-associated viral vector, ora combination thereof.

In an additional embodiment of the present invention there is a methodto prevent breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; identifying in thesample an A908G mutation in a nucleic acid sequence of estrogen receptoralpha; and correcting the A908G mutation, wherein the correction resultsin the prevention of the breast cancer. In a specific embodiment, thebreast sample is from a premalignant lesion of the breast. In anotherspecific embodiment, the correction step comprises administering anestrogen receptor alpha nucleic acid sequence comprising a G atnucleotide 908 to a cell comprising an estrogen receptor alpha nucleicacid sequence containing the A908G mutation.

In an additional embodiment of the present invention there is a methodto treat breast cancer in an individual, wherein an estrogen receptoralpha nucleic acid sequence in a breast cell of the individual has anA908G mutation, comprising the step of administering to the cell anestrogen receptor alpha nucleic acid sequence comprising a G atnucleotide 908.

In another embodiment of the present invention there is a method toprevent breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; identifying in thesample an arginine at amino acid residue 303 in an amino acid sequenceof estrogen receptor alpha; and administering to the individual an aminoacid sequence of estrogen receptor alpha comprising a lysine at aminoacid residue 303, wherein the administration results in the preventionof the breast cancer. In a specific embodiment, the breast sample isfrom a premalignant lesion of the breast.

In an object of the present invention there is a method of identifying amodulator of an estrogen receptor alpha K303R polypeptide, comprisingproviding a candidate modulator; admixing the candidate modulator withan isolated compound or cell, or a suitable experimental animal;measuring one or more characteristics of the compound, cell or animal;and comparing the characteristic measured with the characteristic of thecompound, cell or animal in the absence of the candidate modulator,wherein a difference between the measured characteristics indicates thatthe candidate modulator is the modulator of the compound, cell oranimal.

In another object of the present invention, there is a method ofscreening for a modulator of an estrogen receptor alpha polypeptidecomprising a K303R substitution, comprising introducing to a cell avector comprising a nucleic acid sequence which encodes the estrogenreceptor alpha K303R polypeptide; a vector comprising at least oneestrogen-responsive regulatory element operatively linked to a reporterpolynucleotide; and a test agent; and assaying expression of thereporter polynucleotide in the presence of the test agent, wherein thetest agent is the modulator when the reporter polynucleotide expressionchanges in the presence of the test agent. In a specific embodiment, atleast one of the vectors is transiently transfected into the cell. Inanother specific embodiment, at least one of the vectors is stablytransfected into the cell. In an additional embodiment, when expressionof the reporter polynucleotide is upregulated, the modulator is anagonist. In an additional embodiment, when expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In afurther specific embodiment, when the expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In aspecific embodiment, the cell is a mammalian cell. In a further specificembodiment, the mammalian cell is selected from the group consisting ofCHO, HepG2, HeLa, COS-1, MCF-7, MDA-MB-231, T47D, ZR-75, MDA-MB-435,BT-20, MDA-MB-468, and HEC-1. In an additional specific embodiment, theestrogen-responsive regulatory element is selected from the groupconsisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49;SEQ ID NO:22; SEQ ID NO:26, and SEQ ID NO:8. In an additional specificembodiment, the reporter polynucleotide is luciferase, chloramphenicolacetyltransferase, renilla or β-galactosidase. In a specific embodiment,there is a method of treating breast cancer in an individual comprisingthe step of administering the antagonist to the individual.

In another object of the present invention, there is a method ofidentifying a polypeptide which interacts with an estrogen receptoralpha polypeptide comprising a K303R substitution, comprisingintroducing to a cell, a vector comprising a polynucleotide whichencodes a chimeric polypeptide comprising the estrogen receptor alphaK303R polypeptide and a DNA binding domain; introducing to the cell, avector comprising a polynucleotide which encodes a chimeric polypeptidecomprising a candidate polypeptide and a DNA activation domain; andassaying for an interaction between the DNA binding domain and the DNAactivation domain, wherein when the interaction occurs, the candidatepolypeptide is the polypeptide which interacts with the estrogenreceptor alpha K303R polypeptide. In a specific embodiment, thepolypeptide which interacts with the estrogen receptor alpha K303Rpolypeptide is an antagonist of the estrogen receptor alpha K303Rpolypeptide. In a specific embodiment, the interaction is assayed byassaying for a change in expression of a reporter sequence. In aspecific embodiment, the cell is a yeast cell. In another specificembodiment, the cell is a mammalian cell. In a further specificembodiment, the DNA activation domain and the DNA binding domain arefrom GAL4 or LexA. In an additional specific embodiment, the reportersequence is selected from the group consisting of β-galactosidase,luciferase, chloramphenicol acetyltransferase, and renilla. In aspecific embodiment, there is a method of treating an individual forbreast cancer, comprising administering the antagonist to theindividual.

In another object of the present invention, there is a method ofidentifying a peptide which interacts with an estrogen receptor alphaK303R polypeptide, comprising obtaining an estrogen receptor alpha K303Rpolypeptide having an affinity tag and a label; introducing thepolypeptide to a substrate comprising a plurality of bacteriophage,wherein the bacteriophage produce a candidate peptide; and determiningbinding of the polypeptide with the candidate peptide, wherein when thepolypeptide binds the candidate peptide, the candidate peptide is theinteracting peptide. In a specific embodiment, the label is a colorlabel, a fluorescence label, or a radioactive label. In another specificembodiment, the affinity tag is biotin, GST, histidine, myc, orcalmodulin-binding protein.

In an additional object of the present invention, there is a method ofidentifying a compound for the treatment of breast cancer associatedwith an estrogen receptor alpha K303R polypeptide, comprising the stepsof obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity. In a specificembodiment, the compound having the activity is an antagonist of theestrogen receptor alpha K303R polypeptide. In a specific embodiment, themethod further comprises dispersing the compound in a pharmaceuticalcarrier; and administering a therapeutically effective amount of thecompound in the carrier to an individual having the breast cancer.

Another object of the present invention is the compound obtained by themethod of identifying a compound for the treatment of breast cancerassociated with an estrogen receptor alpha K303R polypeptide, comprisingthe steps of obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity.

An additional object of the present invention is a pharmacologicallyacceptable composition comprising the compound obtained by the method ofidentifying a compound for the treatment of breast cancer associatedwith an estrogen receptor alpha K303R polypeptide, comprising the stepsof obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity; and a pharmaceuticalcarrier.

III. Estrogen Receptor Alpha

Estrogen, mediated through the estrogen receptor (ER), plays a majorrole in regulating the growth and differentiation of normal breastepithelium (Pike et al., 1993; Henderson et al., 1988). It stimulatescell proliferation and regulates the expression of other genes,including the progesterone receptor (PgR). PgR then mediates themitogenic effect of progesterone, further stimulating proliferation(Pike et al., 1993; Henderson et al., 1988). Several studies haveassessed ER expression in normal breast epithelium and premalignantlesions. Studies of normal terminal duct lobular units (TDLUs) reportedthat nearly all (over 90%) express ER, but in a minority (averagingabout 30%) of cells for all ages combined (Schmitt, 1995; Mohsin et al.,2000; Allegra et al., 1979; Peterson et al., 1986; Ricketts et al.,1991). In premenopausal women, the average proportion of ER-positivecells in TDLUs is somewhat lower (about 20%), and varies with themenstrual cycle, being twice as high during the follicular as the lutealphase (Ricketts et al., 1991). Proliferation in TDLUs peaks during theluteal phase (Potten et al., 1988), suggesting that the normal mitogeniceffect of estrogen may be partially delayed or indirect and mediated bydownstream interactions such as that between progesterone and PgR. Inpostmenopausal women, the average proportion of ER-positive cells inTDLUs is relatively high (about 50%) and stable in the absence ofhormone replacement therapy (Mohsin et al., 2000). Very little is knowabout ER expression in ULs, although one preliminary study reported thatvirtually all expressed the receptor in over 90% of cells (Mohsin etal., 2000). A few studies have evaluated ER in ADH and collectivelyagreed that nearly all lesions express very high levels in nearly allcells (Schmitt, 1995; Mohsin et al., 2000; Barnes and Masood, 1990).Many studies have evaluated ER in DCIS and, on average, about 75% of allcases expressed the receptor (Mohsin et al., 2000; Zafrani et al., 1994;Albonico et al., 1996; Berardo et al., 1996; Barnes and Masood, 1990;Helin et al., 1989; Giri et al., 1989; Chaudhuri et al., 1993; Poller etal., 1993; Pallis et al., 1992; Leal et al., 1995; Karayiannakis et al.,1996; Bose et al., 1996). Expression varied with histologicaldifferentiation, being highest in non-comedo (non-mammary ductal)lesions, where up to 100% showed expression in over 90% of cells, andlowest in comedo lesions, where only about 30% showed expression in aminority of cells. ER was not expressed in about 25% of DCIS and thesewere predominately high-grade comedo lesions. Over 90% of LCIS expressedhigh levels of ER in nearly all cells (Fisher et al., 1996; Rudas etal., 1997; Querzoli et al., 1998; Libby et al., 1998; Giri et al., 1989;Pallis et al., 1992; Paertschuk et al., 1990), which is similar in ALHin a specific embodiment.

Prolonged estrogen exposure is an important risk factor for developingIBC, perhaps by allowing random genetic alterations to accumulate innormal cells stimulated to proliferate (Henderson et al. 1988), whichmay also be true for cells in premalignant lesions. The very high levelsof ER observed in nearly all premalignant lesions (FIG. 1) maycontribute to their increased proliferation relative to normal cells byallowing them to respond more effectively to any level of estrogen, eventhe low concentrations seen in postmenopausal women (Mohsin et al.,2000). FIG. 1 illustrates examples of typical estrogen receptorexpression in premalignant breast lesions as assessed byimmunohistochemistry (small dark nuclei are ER-positive cells). Terminalduct lobular units (TDLUs) in premenopausal (pre) women usually containrelatively few ER positive cells. In contrast, the majority of cells inTDLUs of postmenopausal (post) express ER. Most premalignant breastlesions show very high levels of ER in nearly all cells, includingunfolded lobules (Uls), a typical ductal hyperplasias (ADHs), low grade“non-comedo” ductal carcinoma in situ (ncDCIS), a typical lobularhyperplasias (ALHs), and lobular carcinoma in situ (LCIS). The onlysignificant exception is high grade “comedo” DCIS (cDCIS), which oftenshow low or no ER expression.

In addition to increased levels of expression, there may be otheralterations of ER resulting in increased growth in premalignant lesions.For example, in one recent study (Mohsin et al., 2000), proliferationwas measured in TDLUs and premalignant lesions from the same breasts ina large number of patients stratified by menopausal status.Proliferation rates in TDLUs were nearly 3-fold lower in postmenopausalcompared to premenopausal women, consistent with the expected mitogeniceffect of estrogen and progesterone in normal cells. In contrast, thedifference in proliferation in premalignant lesions stratified bymenopausal status was less than half that of normal cells, suggestingthat the hormonal regulation of proliferation in these lesions, in aspecific embodiment, is fundamentally abnormal. It is an object of thepresent invention to diagnose such an abnormality by identifying anA908G mutation in estrogen receptor alpha nucleic acid sequence or aK303R substitution in the amino acid sequence.

IV. Premalignant Lesions of the Breast

Premalignant lesions of the breast are very common, and they are beingdiagnosed more frequently due to increasing public awareness andscreening mammography. They are currently defined by their histologicalfeatures and their prognosis is imprecisely estimated based on indirectepidemiological evidence (Page and Dupont, 1993). While lesions withinspecific categories look alike histologically, there must be underlyingbiological differences causing a subset to progress to IBC. Studiesidentifying biological prognostic factors in premalignant disease arebeginning to emerge (see discussions in Page and Jensen, 1994; Page,1995; Page et al., 1998; Lakhani, 1999). The histopathologicalcharacteristics and anatomic markers associated with premalignantlesions are well known in the art (Cardiff et al., 1977; Bocker, 1997;Page and Dupont, 1990; Stoll, 1999; Lishman and Lakhani, 1999, each ofwhich are incorporated by reference herein in their entirety).

For example, preliminary results from two recent studies suggest thatincreased levels of ER in normal breast epithelium (Kahn et al., 1998)and certain premalignant lesions (UL, ADH, DCIS) (Mohsin et al., 2000)may be associated with a slightly elevated (2-to-3-fold) risk ofdeveloping IBC, and assessing ER status may eventually be important inclinical management. Its most promising role may be in identifyingpatients with high-risk premalignant lesions who might benefit fromhormonal therapy. In the recent NSABP P-1 chemoprevention clinical trial(Fisher et al., 1998), patients with a history of ADH receivingtamoxifen experienced a dramatic decrease (85%) in breast cancerincidence. Nearly all ADH express very high levels of ER, suggestingthat highly ER positive premalignant lesions may be particularlysusceptible to hormonal therapy. The success of this trial isproof-of-principle that targeting biological alterations in premalignantdisease is a rational strategy for the chemoprevention of breast cancer.

Even though microscopic in size, all types of premalignant breastlesions are tumors which expand terminal duct lobular units (TDLUs) andproximal ducts to many times their normal size. Many studies, using avariety of techniques, have measured the magnitude of proliferation inTDLUs and premalignant lesions (Table 1).

TABLE 1 Growth (proliferation and apoptosis) in premalignant breastlesions. TDLU UL ADH DCIS ALH LCIS Average % Proliferation   2% 5% 5%15% “low” 2% Average % Apoptosis 0.6% “low” .03  5% “low” “low”Abbreviations: TDLUs = terminal duct lobular units. ULs = unfoldedlobules. ADH = atypical ductal hyperplasia. DCIS = ductal carcinoma insitu. ALH = atypical lobular hyperplasia. LCIS = lobular carcinoma insitu.

Proliferation in TDLUs averaged only about 2% overall (Meyer, 1977;Ferguson and Anderson, 1981; Joshi et al., 1986; Longacre and Bartow,1986; Russo et al., 1987; Going et al., 1988; Potten et al., 1988; Kamelet al., 1989; Schmitt, 1995; Visscher et al., 1996; Mohsin et al.,2000). In premenopausal women the rate fluctuates with the menstrualcycle and is two-fold higher in the luteal than the follicular phase(Potten et al., 1988). The association between hormonal status andproliferation emphasizes the importance of estrogen and progesterone asmitogens for normal breast epithelium (Pike et al., 1993). Proliferationhas not been evaluated in unfolded lobules (ULs) with the exception ofone preliminary study reporting an average rate of about 5%, which isstill 2-to-3-fold higher than in normal TDLUs (Mohsin et al., 2000).Studies of ADH also observed rates averaging about 5% (Mohsin et al.,2000; De Potter et al., 1987; Hoshi et al., 1995). Proliferation hasbeen studied more extensively in DCIS than any other type ofpremalignant lesion (Mohsin et al., 2000; Meyer, 1986; Locker et al.,1990; Poller et al., 1994; Bobrow et al., 1994; Zafrani et al., 1994;Albonico et al., 1996; Berardo et al., 1996). Rates averaged about 5% inhistologically low-grade “non-comedo” ductal carcinoma in situ (DCIS)compared to 20% in high-grade “comedo” lesions. The wide-spread practiceof dichotomizing DCIS into non-comedo and comedo subtypes is misleadingin the sense that, similar to invasive breast cancer (IBC), DCIS showstremendous histological diversity along a continuum ranging from verywell to very poorly differentiated, and grading systems have beendeveloped which more accurately convey this diversity (Berardo et al.,1996). Proliferation is proportional to differentiation along thiscontinuum, with rates averaging as low as 1% in the lowest grade to morethan 70% in the highest grade lesions (Bobrow et al., 1994; Berardo etal., 1996). Proliferation has not been formally studied in ALH but isprobably similar to LCIS where the reported average is about 2% (Fisheret al., 1996; Rudas et al., 1997; Querzoli et al., 1998; Libby et al.,1998).

The overall growth of premalignant breast lesions can be viewedsimplistically as a balance between cell proliferation and cell death.On average, the cells in all types of premalignant lesions proliferatefaster than normal cells in TDLUs, contributing to their positive growthimbalance. Much less is known about cell death in this setting (Table1). One preliminary study reported significantly lower rates ofapoptosis in a typical ductal hyperplasia (ADH) (0.3%) compared to TDLUs(0.6%) in the same breasts, suggesting that the growth of ADH may be theresult of both increased proliferation and decreased cell death comparedto normal cells (Prosser et al., 1997). However, a few studies havereported rates of apoptosis in DCIS that are much higher (up to 10-fold)than typically seen in normal cells (Prosser et al., 1997; Bodis et al.,1996; Ham et al., 1997), yet DCIS have a profound positive growthimbalance, suggesting that the relationship between cell proliferationand death may not always be accurately portrayed by the static methodsused to measure these dynamic processes. Like proliferation, apoptosisseems to vary with histological differentiation in DCIS, being muchlower in non-comedo (averaging 0.7%) than comedo (averaging 5.6%)lesions (Prosser et al., 1997). Disturbances of the equilibrium betweencell proliferation and death probably result from alterations of severalnormal growth-regulating mechanisms, including those involving sexhormones, oncogenes, tumor suppressor genes, and many other genetic andepigenetic abnormalities.

V. Laser Capture Microdissection

Developments in gene sequencing and amplification techniques, amongothers, now allow scientists to extract DNA or RNA from tissue biopsiesand cytological smears for pinpoint molecular analysis, such as a pointmutation in a nucleic acid sequence. The efficacy of these sophisticatedgenetic testing methods, however, depends on the purity and precision ofthe cell populations being analyzed. Simply homogenizing the biopsysample results in an impure combination of healthy and diseased tissue.Using mechanical tools to manually separate cells of interest from thehistologic section is time-consuming and extremely labor-intensive. Noneof these methods offers the ease, precision and efficiency necessary formodem molecular diagnosis.

The process of laser capture microdissection (LCM) circumvents manyproblems in the art regarding accuracy, efficiency and purity. A laserbeam focally activates a special transfer film which bonds specificallyto cells identified and targeted by microscopy within the tissuesection. The transfer film with the bonded cells is then lifted off thethin tissue section, leaving all unwanted cells behind (which wouldcontaminate the molecular purity of subsequent analysis). Thetransparent transfer film is applied to the surface of the tissuesection. Under the microscope, the diagnostic pathologist or researcherviews the thin tissue section through the glass slide on which it ismounted and chooses microscopic clusters of cells to study. When thecells of choice are in the center of the field of view, the operatorpushes a button which activates a near IR laser diode integral with themicroscope optics. The pulsed laser beam activates a precise spot on thetransfer film immediately above the cells of interest. At this preciselocation the film melts and fuses with the underlying cells of choice.When the film is removed, the chosen cell(s) are tightly held within thefocally expanded polymer, while the rest of the tissue is left behind.This allows multiple homogeneous samples within the tissue section orcytological preparation to be targeted and pooled for extraction ofmolecules and analysis.

In a commercial system, such as with the instruments and methods ofArcturus (Mountain View, Calif.) (http://www.arctur.com/), the film ispermanently bonded to the underside of a transparent vial cap. Amechanical arm precisely positions the transfer surface onto the tissue.The microscope focuses the laser beam to discrete sizes (presentlyeither 30 or 60 micron diameters), delivering precise pulsed doses tothe targeted film. Targeted cells are transferred to the cap surface,and the cap is placed directly onto a vial for molecular processing. Thesize of the targeting pulses is selected by the operator. The cellsadherent to the film retain their morphologic features, and the operatorcan verify that the correct cells have been procured.

Examples of LCM with Breast Tissue include those available athttp://www.arctur.com/technology/lcm_examples/ex_breast.html.

Methods regarding the specific preparations and techniques associatedwith LCM are well known in the art and are provided at(http://www.arctur.com/technology/protocols.html), including:Paraffin-Embedded Tissue, Frozen Tissue, White Blood Cell Cytospin,De-Paraffinization of Tissue Sections, Hematoxylin and Eosin Staining,Immunohistochemical Staining (IHC), Intercalator Dye Staining(Fluorescence), Methyl Green Staining, Nuclear Fast Red Staining, andToluidine Blue O Staining.

An example of Laser Capture Microdissection steps, particularly for usewith Acturus instruments, includes the following:

1. Prepare. Follow routine protocols for preparing a tissue or smear ona standard microscope slide. Apply a Prep Strip™ to flatten the tissueand remove loose debris prior to LCM.

2. Place. Place a CapSure™ HS onto the tissue in the area of interest.The CapSure™ HS is custom designed to keep the transfer film out ofcontact with the tissue.

3. Capture. Pulse the low power infrared laser. The laser activates thetransfer film which then expands down into contact with the tissue. Thedesired cell(s) adhere to the CapSure™ HS transfer film.

4. Microdissect. Lift the CapSure™ HS film carrier, with the desiredcell(s) attached to the film surface. The surrounding tissue remainsintact.

5. Extract. Snap the ExtracSure™ onto the CapSure™ HS. The ExtracSure™is designed to accept low volumes of digestion buffer while sealing outany non-selected material from the captured cells. Pipette theextraction buffer directly into the digestion well of the ExtracSure™.Place a microcentrifuge tube on top.

6. Analyze. Invert the microcentrifuge tube. After centrifuging, thelysate will be at the bottom of the tube. The cell contents, DNA, RNA orprotein, are ready for subsequent molecular analysis.

VI. Mismatch Oligonucleotide Mutation Detection

A skilled artisan recognizes that one method to identify a pointmutation in a nucleic acid sequence is by mismatch oligonucleotidemutation detection, also referred to by other names such asoligonucleotide mismatch detection. In a specific embodiment, a nucleicacid sequence comprising the site to be assayed for the mutation isamplified from a sample, such as by polymerase chain reaction, and amutation is detected with mutation-specific oligonucleotide probehybridization of Southern or slot blots, or a combination thereof.

In a specific embodiment of the present invention, an A908G mutation inestrogen receptor alpha nucleic acid sequence is identified by methodsand/or kits employing oligonucleotide mismatch detection.

VII. Single-Strand Comformation Polymorphism

Single-strand conformation polymorphism (SSCP) (Orita et al., 1989)facilitates detection of polymorphisms, such as single base pairtransitions, through mobility shift analysis on a neutral polyacrylamidegel by methods well known in the art. In specific embodiments, themethod is subsequent to polymerase chain reaction or restriction enzymedigestion, either of which is followed by denaturation for separation ofthe strands. The single stranded species are transferred onto a supportsuch as a nylon membrane, and the mobility shift is detected byhybridization with a nick-translated DNA fragment or with RNA. Inalternative embodiments, the single stranded product is itself labeled,such as with radioactivity, for identification. Samples manifestingmigration shifts in SSCP gels in a specific embodiment are analyzedfurther by other well known methods, such as by DNA sequencing.

In a specific embodiment of the present invention, an A908G mutation inestrogen receptor alpha nucleic acid sequence is identified by methodsand/or kits employing single-strand conformation polymorphism.

VIII. Site-Directed Mutagenesis

Structure-guided site-specific mutagenesis represents a powerful toolfor the dissection and engineering of protein-ligand interactions(Wells, 1996, Braisted et al, 1996). The technique provides for thepreparation and testing of sequence variants by introducing one or morenucleotide sequence changes into a selected DNA.

Site-specific mutagenesis uses specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent, unmodified nucleotides. In this way, a primersequence is provided with sufficient size and complexity to form astable duplex on both sides of the deletion junction being traversed. Aprimer of about 17 to 25 nucleotides in length is preferred, with about5 to 10 residues on both sides of the junction of the sequence beingaltered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage vectors are commercially available and their use is generally wellknown to those skilled in the art. Double-stranded plasmids are alsoroutinely employed in site-directed mutagenesis, which eliminates thestep of transferring the gene of interest from a phage to a plasmid.

In general, one first obtains a single-stranded vector, or melts twostrands of a double-stranded vector, which includes within its sequencea DNA sequence encoding the desired protein or genetic element. Anoligonucleotide primer bearing the desired mutated sequence,synthetically prepared, is then annealed with the single-stranded DNApreparation, taking into account the degree of mismatch when selectinghybridization conditions. The hybridized product is subjected to DNApolymerizing enzymes such as E. coli polymerase I (Klenow fragment) inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed, wherein one strand encodes the originalnon-mutated sequence, and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells, such as E. coli cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

Comprehensive information on the functional significance and informationcontent of a given residue of protein can best be obtained by saturationmutagenesis in which all 19 amino acid substitutions are examined. Theshortcoming of this approach is that the logistics of multiresiduesaturation mutagenesis are daunting (Warren et al., 1996, Brown et al.,1996; Zeng et al., 1996; Burton and Barbas, 1994; Yelton et al., 1995;Jackson et al., 1995; Short et al., 1995; Wong et al., 1996; Hilton etal., 1996). Hundreds, and possibly even thousands, of site specificmutants must be studied. However, improved techniques make productionand rapid screening of mutants much more straightforward. See also, U.S.Pat. Nos. 5,798,208 and 5,830,650, for a description of “walk-through”mutagenesis.

Other methods of site-directed mutagenesis are disclosed in U.S. Pat.Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377;and 5,789,166.

IX. Nucleic Acid Detection

In addition to their use in directing the expression of estrogenreceptor alpha wildtype or mutant proteins, polypeptides and/orpeptides, the nucleic acid sequences disclosed herein have a variety ofother uses. For example, they have utility as probes or primers forembodiments involving nucleic acid hybridization.

A. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

B. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to estrogen receptor alpha wildtype or mutant arecontacted with the template nucleic acid under conditions that permitselective hybridization. Depending upon the desired application, highstringency hybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al, 1990, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known and described in Sambrook et al., 1989.Alternative methods for reverse transcription utilize thermostable DNApolymerases. These methods are described in WO 90/07641. Polymerasechain reaction methodologies are well known in the art. Representativemethods of RT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assy (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). Davey et al., European Application No. 329 822 disclose anucleic acid amplification process involving cyclically synthesizingsingle-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA),which may be used in accordance with the present invention.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoterregion/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme is not cyclic, i.e., new templates are not produced from theresultant RNA transcripts. Other amplification methods include “race”and “one-sided PCR” (Frohman, 1990; Ohara et al., 1989).

C. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art. See Sambrook etal., 1989. One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

D. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion orsubstititution mutations that may be used in the practice of the presentinvention are disclosed in U.S. Pat. Nos. 5,849,483, 5,851,770,5,866,337, 5,925,525 and 5,928,870, each of which is incorporated hereinby reference in its entirety.

E. Kits

All the essential materials and/or reagents required for detectingestrogen receptor alpha wildtype or mutant sequences in a sample may beassembled together in a kit. This generally will comprise a probe orprimers designed to hybridize specifically to individual nucleic acidsof interest in the practice of the present invention, including estrogenreceptor alpha wildtype or mutant sequences. Also included may beenzymes suitable for amplifying nucleic acids, including variouspolymerases (reverse transcriptase, Taq, etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits may also include enzymes and other reagents suitable fordetection of specific nucleic acids or amplification products. Such kitsgenerally will comprise, in suitable means, distinct containers for eachindividual reagent or enzyme as well as for each probe or primer pair.

X. Estrogen Receptor α Nucleic Acids

In a preferred embodiment, an estrogen receptor alpha nucleic acidsequence of the present invention contains an A908G mutation.

In specific embodiments, examples of the estrogen receptor alpha nucleicacid sequences which may include the A908G mutation includeNM_(—)000125.1 (SEQ ID NO:1); AF242866 (SEQ ID NO:2); AF123496.1 (SEQ IDNO:3); AF120105 (SEQ ID NO:4); U47678.1 (SEQ ID NO:5); M12674.1 (SEQ IDNO:6); X03635.1 (SEQ ID NO:7); AF309825 (SEQ ID NO:19); AF061181 (SEQ IDNO:20); AF184588 (SEQ ID NO:21); AF181077 (SEQ ID NO:23); Z37167 (SEQ IDNO:24); AF173235 (SEQ ID NO:25); X90668 (SEQ ID NO:27); and AK025747(SEQ ID NO:28). In other specific embodiments, examples of the estrogenreceptor alpha amino acid sequences which may include the K303Rsubstitution include NP_(—)000116.1 (SEQ ID NO:9); AAF65451.1 (SEQ IDNO:10); AAD23565.1 (SEQ ID NO:11); AAB00115.1 (SEQ ID NO:12); AAA52399.1(SEQ ID NO:13); CAA27284.1 (SEQ ID NO:14); AAF00503.1 (SEQ ID NO:29);AAD53956.1 (SEQ ID NO:30); CAA85524.1 (SEQ ID NO:31); and BAB15231.1(SEQ ID NO:32).

The term “estrogen receptor alpha wildtype or mutant sequence” as usedherein refers respectively to the estrogen receptor alpha wildtypesequence or to a mutant sequence, wherein the mutant sequence comprisesan A908G mutation.

A. Nucleic Acids and Uses Thereof

Certain aspects of the present invention concern at least one estrogenreceptor alpha wildtype and/or mutant nucleic acid. In certain aspects,the at least one estrogen receptor alpha wildtype and/or mutant nucleicacid comprises a wild-type or mutant estrogen receptor alpha wildtypeand/or mutant nucleic acid. In certain aspects, the estrogen receptoralpha wildtype and/or mutant nucleic acid comprises at least onetranscribed nucleic acid. In particular aspects, the estrogen receptoralpha wildtype and/or mutant nucleic acid encodes at least one estrogenreceptor alpha wildtype and/or mutant protein, polypeptide or peptide,or biologically functional equivalent thereof. In other aspects, theestrogen receptor alpha wildtype and/or mutant nucleic acid comprises atleast one nucleic acid segment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:28, or at least one biologically functional equivalentthereof.

The present invention also concerns the isolation or creation of atleast one recombinant construct or at least one recombinant host cellthrough the application of recombinant nucleic acid technology known tothose of skill in the art or as described herein. The recombinantconstruct or host cell may comprise at least one estrogen receptor alphawildtype or mutant nucleic acid, and may express at least one estrogenreceptor alpha wildtype or mutant protein, peptide or peptide, or atleast one biologically functional equivalent thereof.

As used herein “wild-type” refers to the naturally occurring sequence ofa nucleic acid at a genetic locus in the genome of an organism, andsequences transcribed or translated from such a nucleic acid. Thus, theterm “wild-type” also may refer to the amino acid sequence encoded bythe nucleic acid. As a genetic locus may have more than one sequence oralleles in a population of individuals, the term “wild-type” encompassesall such naturally occurring alleles. As used herein the term“polymorphic” means that variation exists (i.e. two or more allelesexist) at a genetic locus in the individuals of a population. As usedherein “mutant” refers to a change in the sequence of a nucleic acid orits encoded protein, polypeptide or peptide that is the result of thehand of man.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemically synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells, such as recombinantDNA vector production in bacteria (see for example, Sambrook et al.1989, incorporated herein by reference).

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al. 1989, incorporatedherein by reference).

The term “nucleic acid” will generally refer to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, comprising at leastone nucleobase, such as, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g. adenine “A,” guanine “G,” thymine “T”and cytosine “C”) or RNA (e.g. A, G, uracil “U” and C). The term“nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule. As used herein,a single stranded nucleic acid may be denoted by the prefix “ss”, adouble stranded nucleic acid by the prefix “ds”, and a triple strandednucleic acid by the prefix “ts.”

Thus, the present invention also encompasses at least one nucleic acidthat is complementary to a estrogen receptor alpha wildtype or mutantnucleic acid. In particular embodiments the invention encompasses atleast one nucleic acid or nucleic acid segment complementary to thesequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, orSEQ ID NO:28. Nucleic acid(s) that are “complementary” or“complement(s)” are those that are capable of base-pairing according tothe standard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein, the term “complementary” or“complement(s)” also refers to nucleic acid(s) that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above. The term “substantially complementary” refers to a nucleicacid comprising at least one sequence of consecutive nucleobases, orsemiconsecutive nucleobases if one or more nucleobase moieties are notpresent in the molecule, are capable of hybridizing to at least onenucleic acid strand or duplex even if less than all nucleobases do notbase pair with a counterpart nucleobase. In certain embodiments, a“substantially complementary” nucleic acid contains at least onesequence in which about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,to about 100%, and any range therein, of the nucleobase sequence iscapable of base-pairing with at least one single or double strandednucleic acid molecule during hybridization. In certain embodiments, theterm “substantially complementary” refers to at least one nucleic acidthat may hybridize to at least one nucleic acid strand or duplex instringent conditions. In certain embodiments, a “partly complementary”nucleic acid comprises at least one sequence that may hybridize in lowstringency conditions to at least one single or double stranded nucleicacid, or contains at least one sequence in which less than about 70% ofthe nucleobase sequence is capable of base-pairing with at least onesingle or double stranded nucleic acid molecule during hybridization.

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “hybridization”, “hybridize(s)” or “capable ofhybridizing” encompasses the terms “stringent condition(s)” or “highstringency” and the terms “low stringency” or “low stringencycondition(s).”

As used herein “stringent condition(s)” or “high stringency” are thosethat allow hybridization between or within one or more nucleic acidstrand(s) containing complementary sequence(s), but precludeshybridization of random sequences. Stringent conditions tolerate little,if any, mismatch between a nucleic acid and a target strand. Suchconditions are well known to those of ordinary skill in the art, and arepreferred for applications requiring high selectivity. Non-limitingapplications include isolating at least one nucleic acid, such as a geneor nucleic acid segment thereof, or detecting at least one specific mRNAtranscript or nucleic acid segment thereof, and the like.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl attemperatures of about 50° C. to about 70° C. It is understood that thetemperature and ionic strength of a desired stringency are determined inpart by the length of the particular nucleic acid(s), the length andnucleobase content of the target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence of formamide,tetramethylammonium chloride or other solvent(s) in the hybridizationmixture. It is generally appreciated that conditions may be renderedmore stringent, such as, for example, the addition of increasing amountsof formamide.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting example only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of the nucleic acid(s) towards targetsequence(s). In a non-limiting example, identification or isolation ofrelated target nucleic acid(s) that do not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

One or more nucleic acid(s) may comprise, or be composed entirely of, atleast one derivative or mimic of at least one nucleobase, a nucleobaselinker moiety and/or backbone moiety that may be present in a naturallyoccurring nucleic acid. As used herein a “derivative” refers to achemically modified or altered form of a naturally occurring molecule,while the terms “mimic” or “analog” refers to a molecule that may or maynot structurally resemble a naturally occurring molecule, but functionssimilarly to the naturally occurring molecule. As used herein, a“moiety” generally refers to a smaller chemical or molecular componentof a larger chemical or molecular structure, and is encompassed by theterm “molecule.”

As used herein a “nucleobase” refers to a naturally occurringheterocyclic base, such as A, T, G, C or U (“naturally occurringnucleobase(s)”), found in at least one naturally occurring nucleic acid(i.e. DNA and RNA), and their naturally or non-naturally occurringderivatives and mimics. Non-limiting examples of nucleobases includepurines and pyrimidines, as well as derivatives and mimics thereof,which generally can form one or more hydrogen bonds (“anneal” or“hybridize”) with at least one naturally occurring nucleobase in mannerthat may substitute for naturally occurring nucleobase pairing (e.g. thehydrogen bonding between A and T, G and C, and A and U).

Nucleobase, nucleoside and nucleotide mimics or derivatives are wellknown in the art, and have been described in exemplary references suchas, for example, Scheit, Nucleotide Analogs (John Wiley, New York,1980), incorporated herein by reference. “Purine” and “pyrimidine”nucleobases encompass naturally occurring purine and pyrimidinenucleobases and also derivatives and mimics thereof, including but notlimited to, those purines and pyrimidines substituted by one or more ofalkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e. fluoro, chloro,bromo, or iodo), thiol, or alkylthiol wherein the alkyl group comprisesof from about 1, about 2, about 3, about 4, about 5, to about 6 carbonatoms. Non-limiting examples of purines and pyrimidines includedeazapurines, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine,8-bromoguanine, 8-chloroguanine, bromothymine, 8-aminoguanine,8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanines,2-aminopurine, 5-ethylcytosine, 5-methylcyosine, 5-bromouracil,5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil,2-methyladenine, methylthioadenine, N,N-diemethyladenine, azaadenines,8-bromoadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine,4-(6-aminohexyl/cytosine), and the like.

As used herein, “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (a “5-carbon sugar”), including but notlimited to deoxyribose, ribose or arabinose, and derivatives or mimicsof 5-carbon sugars. Non-limiting examples of derivatives or mimics of5-carbon sugars include 2′-fluoro-2′-deoxyribose or carbocyclic sugarswhere a carbon is substituted for the oxygen atom in the sugar ring. Byway of non-limiting example, nucleosides comprising purine (i.e. A andG) or 7-deazapurine nucleobases typically covalently attach the 9position of the purine or 7-deazapurine to the 1′-position of a 5-carbonsugar. In another non-limiting example, nucleosides comprisingpyrimidine nucleobases (i.e. C, T or U) typically covalently attach the1 position of the pyrimidine to 1′-position of a 5-carbon sugar(Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco,1992). However, other types of covalent attachments of a nucleobase to anucleobase linker moiety are known in the art, and non-limiting examplesare described herein.

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety” generally used for the covalent attachment of one ormore nucleotides to another molecule or to each other to form one ormore nucleic acids. The “backbone moiety” in naturally occurringnucleotides typically comprises a phosphorus moiety, which is covalentlyattached to a 5-carbon sugar. The attachment of the backbone moietytypically occurs at either the 3′- or 5′-position of the 5-carbon sugar.However, other types of attachments are known in the art, particularlywhen the nucleotide comprises derivatives or mimics of a naturallyoccurring 5-carbon sugar or phosphorus moiety, and non-limiting examplesare described herein.

A non-limiting example of a nucleic acid comprising such nucleoside ornucleotide derivatives and mimics is a “polyether nucleic acid”,described in U.S. Pat. No. 5,908,845, incorporated herein by reference,wherein one or more nucleobases are linked to chiral carbon atoms in apolyether backbone. Another example of a nucleic acid comprisingnucleoside or nucleotide derivatives or mimics is a “peptide nucleicacid”, also known as a “PNA”, “peptide-based nucleic acid mimics” or“PENAMs”, described in U.S. Pat. Nos. 5,786,461, 5891,625, 5,773,571,5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92/20702,each of which is incorporated herein by reference. A peptide nucleicacid generally comprises at least one nucleobase and at least onenucleobase linker moiety that is either not a 5-carbon sugar and/or atleast one backbone moiety that is not a phosphate backbone moiety.Examples of nucleobase linker moieties described for PNAs include azanitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat.No. 5,539,082). Examples of backbone moieties described for PNAs includean aminoethylglycine, polyamide, polyethyl, polythioamide,polysulfinamide or polysulfonamide backbone moiety.

Peptide nucleic acids generally have enhanced sequence specificity,binding properties, and resistance to enzymatic degradation incomparison to molecules such as DNA and RNA (Egholm et al., Nature 1993,365, 566; PCT/EP/01219). In addition, U.S. Pat. Nos. 5,766,855,5,719,262, 5,714,331 and 5,736,336 describe PNAs comprising naturallyand non-naturally occurring nucleobases and alkylamine side chains withfurther improvements in sequence specificity, solubility and bindingaffinity. These properties promote double or triple helix formationbetween a target nucleic acid and the PNA.

U.S. Pat. No. 5,641,625 describes that the binding of a PNA may to atarget sequence has applications the creation of PNA probes tonucleotide sequences, modulating (i.e. enhancing or reducing) geneexpression by binding of a PNA to an expressed nucleotide sequence, andcleavage of specific dsDNA molecules. In certain embodiments, nucleicacid analogues such as one or more peptide nucleic acids may be used toinhibit nucleic acid amplification, such as in PCR, to reduce falsepositives and discriminate between single base mutants, as described inU.S. Pat. Ser. No. 5,891,625.

U.S. Pat. No. 5,786,461 describes PNAs with amino acid side chainsattached to the PNA backbone to enhance solubility. The neutrality ofthe PNA backbone may contribute to the thermal stability of PNA/DNA andPNA/RNA duplexes by reducing charge repulsion. The melting temperatureof PNA containing duplexes, or temperature at which the strands of theduplex release into single stranded molecules, has been described asless dependent upon salt concentration.

One method for increasing amount of cellular uptake property of PNAs isto attach a lipophilic group. U.S. application Ser. No. 117,363, filedSep. 3, 1993, describes several alkylamino functionalities and their usein the attachment of such pendant groups to oligonucleosides. U.S.application Ser. No. 07/943,516, filed Sep. 11, 1992, and itscorresponding published PCT application WO 94/06815, describe othernovel amine-containing compounds and their incorporation intooligonucleotides for, inter alia, the purposes of enhancing cellularuptake, increasing lipophilicity, causing greater cellular retention andincreasing the distribution of the compound within the cell.

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives ormimics are well known in the art.

In certain aspect, the present invention concerns at least one nucleicacid that is an isolated nucleic acid. As used herein, the term“isolated nucleic acid” refers to at least one nucleic acid moleculethat has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells,particularly mammalian cells, and more particularly human cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components and macromolecules such as lipids, proteins, smallbiological molecules, and the like. As different species may have a RNAor a DNA containing genome, the term “isolated nucleic acid” encompassesboth the terms “isolated DNA” and “isolated RNA”. Thus, the isolatednucleic acid may comprise a RNA or DNA molecule isolated from, orotherwise free of, the bulk of total RNA, DNA or other nucleic acids ofa particular species. As used herein, an isolated nucleic acid isolatedfrom a particular species is referred to as a “species specific nucleicacid.” When designating a nucleic acid isolated from a particularspecies, such as human, such a type of nucleic acid may be identified bythe name of the species. For example, a nucleic acid isolated from oneor more humans would be an “isolated human nucleic acid”, a nucleic acidisolated from human would be an “isolated human nucleic acid”, and soforth.

Of course, more than one copy of an isolated nucleic acid may beisolated from biological material, or produced in vitro, using standardtechniques that are known to those of skill in the art. In particularembodiments, the isolated nucleic acid is capable of expressing aprotein, polypeptide or peptide that has the K303R substitution. Inother embodiments, the isolated nucleic acid comprises an isolatedestrogen receptor alpha wildtype or mutant nucleic acid sequence.

Herein certain embodiments, a “gene” refers to a nucleic acid that istranscribed. As used herein, a “gene segment” is a nucleic acid segmentof a gene. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. Inparticular embodiments, the gene comprises transcribed sequences thatencode for a protein, polypeptide or peptide. In other particularaspects, the gene comprises an estrogen receptor alpha wildtype ormutant nucleic acid, and/or encodes an estrogen receptor alpha wildtypeor mutant polypeptide or peptide coding sequences. In keeping with theterminology described herein, an “isolated gene” may comprisetranscribed nucleic acid(s), regulatory sequences, coding sequences, orthe like, isolated substantially away from other such sequences, such asother naturally occurring genes, regulatory sequences, polypeptide orpeptide encoding sequences, etc. In this respect, the term “gene” isused for simplicity to refer to a nucleic acid comprising a nucleotidesequence that is transcribed, and the complement thereof. In particularaspects, the transcribed nucleotide sequence comprises at least onefunctional protein, polypeptide and/or peptide encoding unit. As will beunderstood by those in the art, this function term “gene” includes bothgenomic sequences, RNA or cDNA sequences or smaller engineered nucleicacid segments, including nucleic acid segments of a non-transcribed partof a gene, including but not limited to the non-transcribed promoter orenhancer regions of a gene. Smaller engineered gene nucleic acidsegments may express, or may be adapted to express using nucleic acidmanipulation technology, proteins, polypeptides, domains, peptides,fusion proteins, mutants and/or such like.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case the estrogen receptor alpha gene(s)containing the A908G mutation, forms the significant part of the codingregion of the nucleic acid, or that the nucleic acid does not containlarge portions of naturally-occurring coding nucleic acids, such aslarge chromosomal fragments, other functional genes, RNA or cDNA codingregions. Of course, this refers to the nucleic acid as originallyisolated, and does not exclude genes or coding regions later added tothe nucleic acid by the hand of man.

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment”, are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of the estrogen receptor alpha wildtype or mutant peptide orpolypeptide sequence. In a preferred embodiment, the mutant peptide orpolypeptide sequence comprises the K303R substitution. Thus, a “nucleicacid segment” may comprise any part of the estrogen receptor alphawildtype or mutant gene sequence(s), of from about 2 nucleotides to thefull length of the estrogen receptor alpha wildtype or mutant peptide orpolypeptide encoding region. In certain embodiments, the “nucleic acidsegment” encompasses the full length estrogen receptor alpha wildtype ormutant gene(s) sequence. In particular embodiments, the nucleic acidcomprises any part of the SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, orSEQ ID NO:28 sequence(s), of from about 2 nucleotides to the full lengthof the sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:28.

A non-limiting example of the present invention would be the generationof nucleic acid segments of various lengths and sequence composition forprobes and primers based on the sequences disclosed in SEQID NO:1, SEQIDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:28.

The nucleic acid(s) of the present invention, regardless of the lengthof the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). The length overall length may vary considerably betweennucleic acid constructs. Thus, a nucleic acid segment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation or use in the intended recombinant nucleicacid protocol.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, or SEQID NO:28. A nucleic acid construct may be about 3, about 5, about 8,about 10 to about 14, or about 15, about 20, about 30, about 40, about50, about 100, about 200, about 500, about 1,000, about 2,000, about3,000, about 5,000, about 10,000, about 15,000, about 20,000, about30,000, about 50,000, about 100,000, about 250,000, about 500,000, about750,000, to about 1,000,000 nucleotides in length, as well as constructsof greater size, up to and including chromosomal sizes (including allintermediate lengths and intermediate ranges), given the advent ofnucleic acids constructs such as a yeast artificial chromosome are knownto those of ordinary skill in the art. It will be readily understoodthat “intermediate lengths” and “intermediate ranges”, as used herein,means any length or range including or between the quoted values (i.e.all integers including and between such values). Non-limiting examplesof intermediate lengths include about 11, about 12, about 13, about 16,about 17, about 18, about 19, etc.; about 21, about 22, about 23, etc.;about 31, about 32, etc.; about 51, about 52, about 53, etc.; about 101,about 102, about 103, etc.; about 151, about 152, about 153, etc.; about1,001, about 1002, etc,; about 50,001, about 50,002, etc; about 750,001,about 750,002, etc.; about 1,000,001, about 1,000,002, etc. Non-limitingexamples of intermediate ranges include about 3 to about 32, about 150to about 500,001, about 3,032 to about 7,145, about 5,000 to about15,000, about 20,007 to about 1,000,003, etc.

In particular embodiments, the invention concerns one or morerecombinant vector(s) comprising nucleic acid sequences that encode anestrogen receptor alpha wildtype or mutant protein, polypeptide orpeptide that includes within its amino acid sequence a contiguous aminoacid sequence in accordance with, or essentially as set forth in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32corresponding to human SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, orSEQ ID NO:28. In other embodiments, the invention concerns recombinantvector(s) comprising nucleic acid sequences that encode a human estrogenreceptor alpha wildtype or mutant protein, polypeptide or peptide thatincludes within its amino acid sequence a contiguous amino acid sequencein accordance with, or essentially as set forth in SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In particularaspects, the recombinant vectors are DNA vectors.

The term “a sequence essentially as set forth in SEQ ID NO:9” means thatthe sequence substantially corresponds to a portion of SEQ ID NO:9 andhas relatively few amino acids that are not identical to, or abiologically functional equivalent of, the amino acids of SEQ ID NO:9.Thus, “a sequence essentially as set forth in SEQ ID NO:1 encompassesnucleic acids, nucleic acid segments, and genes that comprise part orall of the nucleic acid sequences as set forth in SEQ ID NO:1.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, a sequencethat has between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; or even more preferably, between about 91% andabout 99%; of amino acids that are identical or functionally equivalentto the amino acids of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, or SEQ ID NO:32 will be a sequence that is “essentially as setforth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ IDNO:32”, provided the biological activity of the protein, polypeptide orpeptide is maintained.

In certain other embodiments, the invention concerns at least onerecombinant vector that include within its sequence a nucleic acidsequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:27, or SEQ ID NO:28. In particular embodiments, therecombinant vector comprises DNA sequences that encode protein(s),polypeptide(s) or peptide(s) exhibiting estrogen receptor alpha wildtypeor mutant activity.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine and serine, and also refers to codons that encode biologicallyequivalent amino acids, which are well known in the art.

Information on codon usage in a variety of non-human organisms is knownin the art (see for example, Bennetzen and Hall, 1982; Ikemura, 1981a,1981b, 1982; Grantham et al., 1980, 1981; Wada et al., 1990; each ofthese references are incorporated herein by reference in theirentirety). Thus, it is contemplated that codon usage may be optimizedfor other animals, as well as other organisms such as fungi, plants,prokaryotes, virus and the like, as well as organelles that containnucleic acids, such as mitochondria, chloroplasts and the like, based onthe preferred codon usage as would be known to those of ordinary skillin the art.

It will also be understood that amino acid sequences or nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, or various combinationsthereof, and yet still be essentially as set forth in one of thesequences disclosed herein, so long as the sequence meets the criteriaset forth above, including the maintenance of biological protein,polypeptide or peptide activity where expression of a proteinaceouscomposition is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences that may, for example,include various non-coding sequences flanking either of the 5′ and/or 3′portions of the coding region or may include various internal sequences,i.e., introns, which are known to occur within genes.

Excepting intronic and flanking regions, and allowing for the degeneracyof the genetic code, nucleic acid sequences that have between about 70%and about 79%; or more preferably, between about 80% and about 89%; oreven more particularly, between about 90% and about 99%; of nucleotidesthat are identical to the nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:27, or SEQ ID NO:28 will be nucleic acid sequences thatare “essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:28”.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:27, or SEQ ID NO:28, or the amino acid sequences of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32,respectively. Recombinant vectors and isolated nucleic acid segments maytherefore variously include these coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, and they may encode larger polypeptides or peptides thatnevertheless include such coding regions or may encode biologicallyfunctional equivalent proteins, polypeptide or peptides that have mutantamino acids sequences.

The nucleic acids of the present invention encompass biologicallyfunctional equivalent estrogen receptor alpha wildtype or mutantproteins, polypeptides, or peptides. Such sequences may arise as aconsequence of codon redundancy or functional equivalency that are knownto occur naturally within nucleic acid sequences or the proteins,polypeptides or peptides thus encoded. Alternatively, functionallyequivalent proteins, polypeptides or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein, polypeptide or peptide structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced, for example, through theapplication of site-directed mutagenesis techniques as discussed hereinbelow, e.g., to introduce improvements or alterations to theantigenicity of the protein, polypeptide or peptide, or to test mutantsin order to examine estrogen receptor alpha wildtype or mutant protein,polypeptide or peptide activity at the molecular level.

Fusion proteins, polypeptides or peptides may be prepared, e.g., wherethe estrogen receptor alpha wildtype or mutant coding regions arealigned within the same expression unit with other proteins,polypeptides or peptides having desired functions. Non-limiting examplesof such desired functions of expression sequences include purificationor immunodetection purposes for the added expression sequences, e.g.,proteinaceous compositions that may be purified by affinitychromatography or the enzyme labeling of coding regions, respectively.

Encompassed by the invention are nucleic acid sequences encodingrelatively small peptides or fusion peptides, such as, for example,peptides of from about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, about 31, about 32, about 33, about 34, about 35, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 51, about 52, about 53, about 54, about 55, about 56,about 57, about 58, about 59, about 60, about 61, about 62, about 63,about 64, about 65, about 66, about 67, about 68, about 69, about 70,about 71, about 72, about 73, about 74, about 75, about 76, about 77,about 78, about 79, about 80, about 81, about 82, about 83, about 84,about 85, about 86, about 87, about 88, about 89, about 90, about 91,about 92, about 93, about 94, about 95, about 96, about 97, about 98,about 99, to about 100 amino acids in length, or more preferably, offrom about 15 to about 30 amino acids in length; as set forth in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, andalso larger polypeptides up to and including proteins corresponding tothe full-length sequences set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, or SEQ ID NO:32.

As used herein an “organism” may be a prokaryote, eukaryote, virus andthe like. As used herein the term “sequence” encompasses both the terms“nucleic acid” and “proteinaceous” or “proteinaceous composition.” Asused herein, the term “proteinaceous composition” encompasses the terms“protein”, “polypeptide” and “peptide.” As used herein “artificialsequence” refers to a sequence of a nucleic acid not derived fromsequence naturally occurring at a genetic locus, as well as the sequenceof any proteins, polypeptides or peptides encoded by such a nucleicacid. A “synthetic sequence”, refers to a nucleic acid or proteinaceouscomposition produced by chemical synthesis in vitro, rather thanenzymatic production in vitro (i.e. an “enzymatically produced”sequence) or biological production in vivo (i.e. a “biologicallyproduced” sequence).

XI. Protein Computer Modeling

To determine whether a mutation would likely produce a protein,polypeptide or peptide with a less exposed site and/or motif, theputative location of the altered, moved or added site and/or sequencecould be determined by comparison of the mutated sequence to that of theunmutated protein, polypeptide or peptide's secondary and tertiarystructure, as determined by such methods known to those of ordinaryskill in the art including, but not limited to, X-ray crystallography,NMR or computer modeling. Computer models of various polypeptide andpeptide structures are also available in the literature or computerdatabases. In a non-limiting example, the Entrez database(http://www.ncbi.nlm.nih.gov/Entrez/) may be used by one of ordinaryskill in the art to identify target sequences and regions formutagenesis. The Entrez database is crosslinked to a database of 3-Dstructures for the identified amino acid sequence, if known. Suchmolecular models may be used to identify sites and/or flanking sequencesin peptides and polypeptides that are more exposed to contact withexternal molecules, (e.g. receptors) than similar sequences embedded inthe interior of the polypeptide or polypeptide. In certain embodiments,when adding at least one site and/or flanking sequence is desirable,regjons of the protein that are more exposed to contact with externalmolecules are preferred as sites to add such a sequence. The mutated orwild-type protein, polypeptide or peptide's structure could bedetermined by X-ray crystallography or NMR directly before use in invitro or in vivo assays, as would be known to one of ordinary skill inthe art.

XII. Prokaryotic Peptide Display

Molecular analysis of naturally occurring and artificial proteinlibraries has been greatly improved by the development of various“display” methodologies. The general scheme behind display techniques isthe advantageous expression of peptides, and their disposition on somebiological surface (phage, cell, etc.). The ability of different versionof the displaying organism to present millions and millions of differentvariants allows the rapid screening of the corresponding library forbiological function.

In U.S. Pat. No. 5,821,047, monovalent phage display is described. Thismethod provides for the selection of novel proteins, and variantsthereof. The method comprises fusing a gene encoding a protein ofinterest to the carboxy terminal domain of the gene III coat protein ofthe filamentous phage M13. The fusion is mutated to form a library ofstructurally related fusion proteins that are expressed in low quantityon the surface of phagemid candidates.

U.S. Pat. No. 5,571,698 describes directed evolution using an M13phagemid system. A protein is expression as a fusion with the M13 geneIII protein. Successive rounds of mutagenesis are performed, each timeselecting for improved biological function, e.g., binding of a proteinto a cognate binding partner.

Heterodimer phage libraries are described in U.S. Pat. No. 5,759,817.Filamentous phage comprising a matrix of cpVIII proteins encapsulating agenome encoding first and second polypeptides of an autogenouslyassembling receptor, such as an antibody, are provided. The receptor issurface-integrated into the phage coat matrix via the cpVIII membraneanchor, presenting the receptor for biological assessment.

Another system, lambdoid phage, also can be used for display purposes.In U.S. Pat. No. 5,672,024, lambdoid phage comprising a matrix ofproteins encapsulating a genome encoding first and second polypeptidesof an autogenously assembling receptor are prepared. Thesurface-integrated receptor is available on the surface on the phage forcharacterization.

Immunoglobulin heavy chain libraries are displayed by phage as describedin U.S. Pat. No. 5,824,520. A single chain antibody library is generatedby creating highly divergent, synthetic hypervariable regions, followedby phage display and selection. The resulting antibodies were used toinhibit intracellular enzyme activity. Another patent describingantibody display is U.S. Pat. No. 5,922,545.

Another example of phage display can be found in U.S. Pat. No.5,780,279. This method provides for the identification and selection ofnovel substrates for enzymes. The method comprises constructing a genefusion comprising DNA encoding a polypeptide fused to a DNA encoding asubstrate peptide, which in turn is fusion to DNA encoding at least aportion of a phage coat protein. The DNA encoding the substrate peptideis mutated at one or more codons, thereby generating a family ofmutants. The fusion protein is expressed on the surface of the phagemidparticle and subjected to chemical or enzymatic modification of thesubstrate peptide. Those phagemid particles that have been modified arethen separated from those that have not.

Bacteria also have been used successfully to display proteins. U.S. Pat.No. 5,348,867, describes expression of proteins on bacterial surfaces.The compositions and methods provide stable, surface-expressedpolypeptide from recombinant gram-negative bacterial cell hosts. Atripartite chimeric gene and its related recombinant vector includeseparate DNA sequences for directing or targeting and translocating adesired gene product from a cell periplasm to the external cell surface.A wide range of polypeptides may be efficiently surface expressed usingthis system. See also, U.S. Pat. Nos. 5,508,192 and 5,866,344.

U.S. Pat. No. 5,500,353 describes another bacterial display system.Bacteria (e.g., Caulobacter) having a S-layer modified such that thebacterium S-layer protein gene contains one or more in-frame fusionscoding for one or more heterologous peptides or polypeptides isdescribed. The proteins are expressed on the surface of the bacterium,which may advantageously be cultured as a film.

XIII. Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active compounds. By creating such analogs, it is possibleto fashion drugs which are more active or stable than the naturalmolecules, which have different susceptibility to alteration or whichmay affect the function of various other molecules. In one approach, onewould generate a three-dimensional structure for the antagonist ofestrogen receptor alpha K303R polypeptide of the invention or a fragmentthereof. This could be accomplished by X-ray crystallography, computermodeling or by a combination of both approaches. An alternative approachinvolves the random replacement of functional groups throughout theestrogen receptor alpha K303R polypeptide, and the resulting affect onfunction determined.

It also is possible to isolate a estrogen receptor alpha K303Rpolypeptide specific antibody, selected by a functional assay, and thensolve its crystal structure. In principle, this approach yields apharmacore upon which subsequent drug design can be based. It ispossible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

Thus, one may design drugs which have enhanced and improved biologicalactivity, for example, anti-breast cancer activity relative to astarting compound. By virtue of the chemical isolation procedures anddescriptions well known in the art, sufficient amounts of the estrogenreceptor alpha K303R polypeptide of the invention can be produced toperform crystallographic studies. In addition, knowledge of the chemicalcharacteristics of these compounds permits computer employed predictionsof structure-function relationships that facilitate drug design.

XIV. Screening for Modulators of the Protein Function

The present invention further comprises methods for identifyingmodulators of the function of an estrogen receptor alpha K303Rpolypeptide. These assays may comprise random screening of largelibraries of candidate substances; alternatively, the assays may be usedto focus on particular classes of compounds selected with an eye towardsstructural attributes that are believed to make them more likely tomodulate the function of estrogen receptor alpha K303R polypeptide.

By fuction, it is meant that one may assay for antagonist and/or agonistactivity of an estrogen receptor alpha K303R polypeptide.

To identify a estrogen receptor alpha K303R polypeptide modulator, onegenerally will determine the function of estrogen receptor alpha K303Rpolypeptide in the presence and absence of the candidate substance, amodulator defined as any substance that alters function. For example, amethod generally comprises:

-   -   (a) providing a candidate modulator;    -   (b) admixing the candidate modulator with an isolated compound        or cell, or a suitable experimental animal;    -   (c) measuring one or more characteristics of the compound, cell        or animal in step (b); and    -   (d) comparing the characteristic measured in step (c) with the        characteristic of the compound, cell or animal in the absence of        said candidate modulator,    -   wherein a difference between the measured characteristics        indicates that said candidate modulator is, indeed, a modulator        of the compound, cell or animal.

Assays may be conducted in cell free systems, in isolated cells, or inorganisms including transgenic animals.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

A. Modulators

As used herein the term “candidate substance” refers to any moleculethat may potentially inhibit or enhance estrogen receptor alpha K303Rpolypeptide activity. The candidate substance may be a protein orfragment thereof, a small molecule, or even a nucleic acid molecule. Itmay prove to be the case that the most useful pharmacological compoundswill be compounds that are structurally related to SERMs. Using leadcompounds to help develop improved compounds is know as “rational drugdesign” and includes not only comparisons with know inhibitors andactivators, but predictions relating to the structure of targetmolecules.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or target compounds. By creating suchanalogs, it is possible to fashion drugs, which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for a target molecule, or a fragment thereof. This could beaccomplished by x-ray crystallography, computer modeling or by acombination of both approaches.

It also is possible to use antibodies to ascertain the structure of atarget compound activator or inhibitor. In principle, this approachyields a pharmacore upon which subsequent drug design can be based. Itis possible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

Candidate compounds may include fragments or parts ofnaturally-occurring compounds, or may be found as active combinations ofknown compounds, which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may be peptide,polypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design startingfrom known inhibitors or stimulators.

Other suitable modulators include antisense molecules, ribozymes, andantibodies (including single chain antibodies), each of which would bespecific for the target molecule. Such compounds are described ingreater detail elsewhere in this document. For example, an antisensemolecule that bound to a translational or transcriptional start site, orsplice junctions, would be ideal candidate inhibitors.

In addition to the modulating compounds initially identified, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the structure of themodulators. Such compounds, which may include peptidomimetics of peptidemodulators, may be used in the same manner as the initial modulators.

An inhibitor according to the present invention may be one which exertsits inhibitory or activating effect upstream, downstream or directly onan estrogen receptor alpha K303R polypeptide. Regardless of the type ofinhibitor or activator identified by the present screening methods, theeffect of the inhibition or activator by such a compound results inreduction in the activity of estrogen receptor alpha K303R polypeptideas a transcription factor as compared to that observed in the absence ofthe added candidate substance.

B. In vitro Assays

A quick, inexpensive and easy assay to run is an in vitro assay. Suchassays generally use isolated molecules, can be run quickly and in largenumbers, thereby increasing the amount of information obtainable in ashort period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

One example of a cell free assay is a binding assay. While not directlyaddressing function, the ability of a modulator to bind to a targetmolecule in a specific fashion is strong evidence of a relatedbiological effect. For example, binding of a molecule to a target may,in and of itself, be inhibitory, due to steric, allosteric orcharge-charge interactions. The target may be either free in solution,fixed to a support, expressed in or on the surface of a cell. Either thetarget or the compound may be labeled, thereby permitting determining ofbinding. Usually, the target will be the labeled species, decreasing thechance that the labeling will interfere with or enhance binding.Competitive binding formats can be performed in which one of the agentsis labeled, and one may measure the amount of free label versus boundlabel to determine the effect on binding.

A technique for high throughput screening of compounds is described inWO 84/03564. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. Bound polypeptide is detected by various methods.

C. In Cyto Assays

The present invention also contemplates the screening of compounds fortheir ability to modulate estrogen receptor alpha K303R polypeptide incells. Various cell lines can be utilized for such screening assays,including cells specifically engineered for this purpose. For example,cells comprising an estrogen receptor alpha K303R polypeptide-expressingvector, a vector comprising an estrogen regulatory element operativelylinked to a reporter polynucleotide, and a compound to be screened arecontemplated.

Depending on the assay, culture may be required. The cell is examinedusing any of a number of different physiologic assays. Alternatively,molecular analysis may be performed, for example, looking at proteinexpression, mRNA expression (including differential display of wholecell or polyA RNA) and others.

D. In vivo Assays

In vivo assays involve the use of various animal models, includingtransgenic animals that have been engineered to have specific defects,or carry markers that can be used to measure the ability of a candidatesubstance to reach and effect different cells within the organism. Dueto their size, ease of handling, and information on their physiology andgenetic make-up, mice are a preferred embodiment, especially fortransgenics. However, other animals are suitable as well, includingrats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs,sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbonsand baboons). Assays for modulators may be conducted using an animalmodel derived from any of these species.

In such assays, one or more candidate substances are administered to ananimal, and the ability of the candidate substance(s) to alter one ormore characteristics, as compared to a similar animal not treated withthe candidate substance(s), identifies a modulator. The characteristicsmay be any of those discussed above with regard to the function of aparticular compound (e.g., enzyme, receptor, hormone) or cell (e.g.,growth, tumorigenicity, survival), or instead a broader indication suchas behavior, anemia, immune response, etc.

The present invention provides methods of screening for a candidatesubstance that antagonizes an estrogen receptor alpha K303R polypeptide.In these embodiments, the present invention is directed to a method fordetermining the ability of a candidate substance to reduce the activityof estrogen receptor alpha K303R polypeptide, generally including thesteps of: administering a candidate substance to the animal; anddetermining the ability of the candidate substance to reduce one or morecharacteristics of estrogen receptor alpha K303R polypeptide.

Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route that could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated routes are systemic intravenous injection,regional administration via blood or lymph supply, or directly to anaffected site.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Also, measuring toxicity and doseresponse can be performed in animals in a more meaningful fashion thanin in vitro or in cyto assays.

XV. Mimetics

The present inventors contemplate that structurally similar compoundsmay be formulated to mimic the key portions of peptide or polypeptidesof the present invention. Such compounds, which may be termedpeptidomimetics, may be used in the same manner as the peptides of theinvention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins. Vita et al. (1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

XVI. Immunodetection Methods

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asestrogen receptor alpha protein or nucleic acid components. The estrogenreceptor alpha antibodies prepared in accordance with the presentinvention may be employed to detect wild-type and/or mutant estrogenreceptor alpha proteins, polypeptides and/or peptides. In specificembodiments, the antibodies detect an acetylated form of estrogenreceptor alpha protein, polypeptide and/or peptide or the antibodiesdetect an A908G estrogen receptor alpha nucleic acid mutation. The useof wild-type and/or mutant estrogen receptor alpha specific antibodiesis contemplated. Some immunodetection methods include enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometricassay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay,and Western blot to mention a few. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle M H and Ben-Zeev O, 1999; Gulbis Band Galand P, 1993; De Jager R et al., 1993; and Nakamura et al., 1987,each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing estrogen receptor alpha protein, polypeptideand/or peptide, and contacting the sample with a first anti-estrogenreceptor alpha antibody in accordance with the present invention, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes.

These methods include methods for purifying wild-type and/or mutantestrogen receptor alpha proteins, polypeptides and/or peptides as may beemployed in purifying wild-type and/or mutant estrogen receptor alphaproteins, polypeptides and/or peptides from patients' samples and/or forpurifying recombinantly expressed wild-type or mutant estrogen receptoralpha proteins, polypeptides and/or peptides. In these instances, theantibody removes the antigenic wild-type and/or mutant estrogen receptoralpha protein, polypeptide and/or peptide component from a sample. Theantibody will preferably be linked to a solid support, such as in theform of a column matrix, and the sample suspected of containing thewild-type or mutant estrogen receptor alpha protein antigenic componentwill be applied to the immobilized antibody. The unwanted componentswill be washed from the column, leaving the antigen immunocomplexed tothe immobilized antibody, which wild-type or mutant estrogen receptoralpha protein antigen is then collected by removing the wild-type ormutant estrogen receptor alpha protein and/or peptide from the column.

The immunobinding methods also include methods for detecting andquantifying the amount of a wild-type or mutant estrogen receptor alphaprotein reactive component in a sample and the detection andquantification of any immune complexes formed during the bindingprocess. Here, one would obtain a sample suspected of containing awild-type or mutant estrogen receptor alpha protein and/or peptide, andcontact the sample with an antibody against wild-type or mutant estrogenreceptor alpha, and then detect and quantify the amount of immunecomplexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing a wild-type or mutant estrogenreceptor alpha protein-specific antigen, such as a breast tissue sectionor specimen, a homogenized breast tissue extract, a breast cell,separated and/or purified forms of any of the above wild-type or mutantestrogen receptor alpha protein-containing compositions, or even anybiological fluid that comes into contact with the breast tissue.Diseases that may be suspected of containing a wild-type or mutantestrogen receptor alpha protein-specific antigen include, but are notlimited to, breast cancer.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any estrogenreceptor alpha protein antigens present. After this time, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or western blot, will generally be washed to remove anynon-specifically bound antibody species, allowing only those antibodiesspecifically bound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The estrogen receptor alpha antibody employed in the detection mayitself be linked to a detectable label, wherein one would then simplydetect this label, thereby allowing the amount of the primary immunecomplexes in the composition to be determined. Alternatively, the firstantibody that becomes bound within the primary immune complexes may bedetected by means of a second binding ligand that has binding affinityfor the antibody. In these cases, the second binding ligand may belinked to a detectable label. The second binding ligand is itself oftenan antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under effective conditions and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousforms of cancer, such as breast cancer. Here, a biological and/orclinical sample suspected of containing a wild-type or mutant estrogenreceptor alpha protein, polypeptide, peptide and/or mutant is used.However, these embodiments also have applications to non-clinicalsamples, such as in the titering of antigen or antibody samples, forexample in the selection of hybridomas.

In the clinical diagnosis and/or monitoring of patients with variousforms of breast cancer, the detection of estrogen receptor alpha mutant,and/or an alteration in the levels of estrogen receptor alpha, incomparison to the levels in a corresponding biological sample from anormal subject is indicative of a patient with cancer, such as breastcancer. However, as is known to those of skill in the art, such aclinical diagnosis would not necessarily be made on the basis of thismethod in isolation. Those of skill in the art are very familiar withdifferentiating between significant differences in types and/or amountsof biomarkers, which represent a positive identification, and/or lowlevel and/or background changes of biomarkers. Indeed, backgroundexpression levels are often used to form a “cut-off” above whichincreased detection will be scored as significant and/or positive.

A. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, the anti-estrogen receptor alpha antibodies ofthe invention are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing the wild-type and/or mutant estrogenreceptor alpha protein antigen, such as a clinical sample, is added tothe wells. After binding and/or washing to remove non-specifically boundimmune complexes, the bound wild-type and/or mutant estrogen receptoralpha protein antigen may be detected. Detection is generally achievedby the addition of another anti-estrogen receptor alpha antibody that islinked to a detectable label. This type of ELISA is a simple “sandwichELISA”. Detection may also be achieved by the addition of a secondanti-estrogen receptor alpha antibody, followed by the addition of athird antibody that has binding affinity for the second antibody, withthe third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing thewild-type and/or mutant estrogen receptor alpha protein antigen areimmobilized onto the well surface and/or then contacted with theanti-estrogen receptor alpha antibodies of the invention. After bindingand/or washing to remove non-specifically bound immune complexes, thebound anti-estrogen receptor alpha antibodies are detected. Where theinitial anti-estrogen receptor alpha antibodies are linked to adetectable label, the immune complexes may be detected directly. Again,the immune complexes may be detected using a second antibody that hasbinding affinity for the first anti-estrogen receptor alpha antibody,with the second antibody being linked to a detectable label.

Another ELISA in which the wild-type and/or mutant estrogen receptoralpha proteins, polypeptides and/or peptides are immobilized, involvesthe use of antibody competition in the detection. In this ELISA, labeledantibodies against wild-type or mutant estrogen receptor alpha proteinare added to the wells, allowed to bind, and/or detected by means oftheir label. The amount of wild-type or mutant estrogen receptor alphaprotein antigen in an unknown sample is then determined by mixing thesample with the labeled antibodies against wild-type and/or mutantestrogen receptor alpha before and/or during incubation with coatedwells. The presence of wild-type and/or mutant estrogen receptor alphaprotein in the sample acts to reduce the amount of antibody againstwild-type or mutant estrogen receptor alpha protein available forbinding to the well and thus reduces the ultimate signal. This is alsoappropriate for detecting antibodies against wild-type or mutantestrogen receptor alpha protein in an unknown sample, where theunlabeled antibodies bind to the antigen-coated wells and also reducesthe amount of antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

B. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). The method ofpreparing tissue blocks from these particulate specimens has beensuccessfully used in previous IHC studies of various prognostic factors,and/or is well known to those of skill in the art (Brown et al., 1990;Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

C. Immunodetection Kits

In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedabove. As the estrogen receptor alpha antibodies are generally used todetect wild-type and/or mutant estrogen receptor alpha proteins,polypeptides and/or peptides, or to detect the A908G mutation inestrogen receptor nucleic acid sequence, the antibodies will preferablybe included in the kit. However, kits including both such components maybe provided. The immunodetection kits will thus comprise, in suitablecontainer means, a first antibody that binds to a wild-type and/ormutant estrogen receptor alpha protein, polypeptide and/or peptide,and/or optionally, an immunodetection reagent and/or further optionally,a wild-type and/or mutant estrogen receptor alpha protein, polypeptideand/or peptide.

In preferred embodiments, monoclonal antibodies will be used. In certainembodiments, the first antibody that binds to the wild-type and/ormutant estrogen receptor alpha protein, polypeptide and/or peptide maybe pre-bound to a solid support, such as a column matrix and/or well ofa microtitre plate.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with and/orlinked to the given antibody. Detectable labels that are associated withand/or attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and/or all such labels may beemployed in connection with the present invention.

The kits may further comprise a suitably aliquoted composition of thewild-type and/or mutant estrogen receptor alpha protein, polypeptideand/or polypeptide, whether labeled and/or unlabeled, as may be used toprepare a standard curve for a detection assay. The kits may containantibody-label conjugates either in fully conjugated form, in the formof intermediates, and/or as separate moieties to be conjugated by theuser of the kit. The components of the kits may be packaged either inaqueous media and/or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe and/or other container means,into which the antibody may be placed, and/or preferably, suitablyaliquoted. Where wild-type and/or mutant estrogen receptor alphaprotein, polypeptide and/or peptide, and/or a second and/or thirdbinding ligand and/or additional component is provided, the kit willalso generally contain a second, third and/or other additional containerinto which this ligand and/or component may be placed. The kits of thepresent invention will also typically include a means for containing theantibody, antigen, and/or any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionand/or blow-molded plastic containers into which the desired vials areretained.

XVII. Two Hybrid Screen

The term “two hybrid screen” as used herein refers to a screen toelucidate or characterize the function of a protein by identifying otherproteins with which it interacts. The protein of unknown function,herein referred to as the “bait” is produced as a chimeric proteinadditionally containing the DNA binding domain of, for example, GAL4.Plasmids containing nucleotide sequences which express this chimericprotein are transformed into yeast cells, which also contain arepresentative plasmid from a library containing the respective GAL4activation domain fused to different nucleotide sequences encodingdifferent potential target proteins. If the bait protein physicallyinteracts with a target protein, the GAL4 activation domain and GAL4 DNAbinding domain are tethered and are thereby able to act conjunctively topromote transcription of a reporter gene. If no interaction occursbetween the bait protein and the potential target protein in aparticular cell, the GAL4 components remain separate and unable topromote reporter gene transcription on their own. One skilled in the artis aware that different reporter genes can be utilized, includingβ-galactosidase, HIS3, ADE2, or URA3. Furthermore, multiple reportersequences, each under the control of a different inducible promoter, canbe utilized within the same cell to indicate interaction of the GAL4components (and thus a specific bait and target protein). A skilledartisan is aware that use of multiple reporter sequences decreases thechances of obtaining false positive candidates. Also, alternativeDNA-binding domain/activation domain components may be used, such asLexA. One skilled in the art is aware that any activation domain may bepaired with any DNA binding domain so long as they are able to generatetransactivation of a reporter gene. Furthermore, a skilled artisan isaware that either of the two components may be of prokaryotic origin, aslong as the other component is present and they jointly allowtransactivation of the reporter gene, as with the LexA system.

Two hybrid experimental reagents and design are well known to thoseskilled in the art (see The Yeast Two-Hybrid System by P. L. Bartel andS. Fields (eds.) (Oxford University Press, 1997), including the mostupdated improvements of the system (Fashena et al., 2000). A skilledartisan is aware of commercially available vectors, such as theMatchmaker™ Systems from Clontech (Palo Alto, Calif.) or the HybriZAP®2.1 Two Hybrid System (Stratagene; La Jolla, Calif.), or vectorsavailable through the research community (Yang et al., 1995; James etal., 1996). In alternative embodiments, organisms other than yeast areused for two-hybrid analysis, such as mammals (Mammalian Two HybridAssay Kit from Stratagene (La Jolla, Calif.)) or E. coli (Hu et al.,2000).

In an alternative embodiment, a two-hybrid system is utilized whereinprotein-protein interactions are detected in a cytoplasmic-based assay.In this embodiment, proteins are expressed in the cytoplasm, whichallows posttranslational modifications to occur and permitstranscriptional activators and inhibitors to be used as bait in thescreen. An example of such a system is the CytoTrap® Two-Hybrid Systemfrom Stratagene™ (La Jolla, Calif.), in which a target protein becomesanchored to a cell membrane of a yeast which contains a temperaturesensitive mutation in the cdc25 gene, the yeast homolog for hSos (aguanyl nucleotide exchange factor). Upon binding of a bait protein tothe target, hSos is localized to the membrane, which allos activation ofRAS by promoting GDP/GTP exchange. RAS then activates a signalingcascade which allows growth at 37° C. of a mutant yeast cdc25H. Vectors(such as pMyr and pSos) and other experimental details are available forthis system to a skilled artisan through Stratagene (La Jolla, Calif.).(See also, for example, U.S. Pat. No. 5,776,689, herein incorporated byreference).

Thus, in accordance with an embodiment of the present invention, thereis a method of screening for a peptide which interacts with ERα K303Rpolypeptide comprising introducing into a cell a first nucleic acidcomprising a DNA segment encoding a test peptide, wherein the testpeptide is fused to a DNA activation domain, and a second nucleic acidcomprising a DNA segment encoding at least part of ERα K303Rpolypeptide, respectively, wherein the at least part of ERα K303Rpolypeptide, respectively, is fused to a DNA binding domain.Subsequently, there is an assay for interaction between the test peptideand the ERα K303R polypeptide or fragment thereof by assaying forinteraction between the DNA activation domain and the DNA bindingdomain. In a preferred embodiment, the assay for interaction between theDNA binding and activation domains is activation of expression ofβ-galactosidase. In an alternative embodiment, the ERα K303R polypeptideis fused to the DNA activation domain and the test peptides are fused tothe DNA binding domain.

XVIII. Cancer

Tumors are notoriously heterogeneous, particularly in advanced stages oftumor progression (Morton et al., 1993; Fidler and Hart, 1982; Nowell,1982; Elder et al., 1989; Bystryn et al., 1985). Although tumor cellswithin a primary tumor or metastasis all may express the same markergene, the level of specific mRNA expression can vary considerably (Elderet al., 1989). It is, in certain instances, necessary to employ adetection system that can cope with an array of heterogeneous markers.In a specific embodiment, a marker for breast cancer comprises an A908Gestrogen receptor alpha nucleic acid sequence or the K303R substitutionto which it corresponds, or both.

Thus, while the present invention exemplifies various tumor suppressorsas a markers, any marker that is correlated with the presence or absenceof cancer may be used in combination with these markers to improve theefficacy of tumor detection and treatment. A marker, as used herein, isany proteinaceous molecule (or corresponding gene) whose production orlack of production is characteristic of a cancer cell. Depending on theparticular set of markers employed in a given analysis, the statisticalanalysis will vary. For example, where a particular combination ofmarkers is highly specific for melanomas or breast cancer, thestatistical significance of a positive result will be high. It may be,however, that such specificity is achieved at the cost of sensitivity,i.e., a negative result may occur even in the presence of melanoma orbreast cancer. By the same token, a different combination may be verysensitive, i.e., few false negatives, but has a lower specificity.

As new markers are identified, different combinations may be developedthat show optimal function with different ethnic groups or sex,different geographic distributions, different stages of disease,different degrees of specificity or different degrees of sensitivity.Marker combinations also may be developed, which are particularlysensitive to the effect of therapeutic regimens on disease progression.Patients may be monitored after surgery, gene therapy, hyperthermia,immunotherapy, cytokine therapy, gene therapy, radiotherapy orchemotherapy, to determine if a specific therapy is effective.

There are many other markers that may be used in combination with these,and other, markers. For example, β-human chorionic gonadotropin (β-HCG)is produced by trophoblastic cells of placenta of pregnant woman and isessential for maintenance of pregnancy at the early stages (Pierce etal., 1981; Talmadge et al., 1984). b-HCG is known to be produced bytrophoblastic or germ cell origin tumors, such as choriocarcinoma ortesticular carcinoma cells (Madersbacher et al., 1994; Cole et al.,1983). Also ectopic expression of b-HCG has been detected by a number ofdifferent immunoassays in various tumors of non-gonadal such as breast,lung, gastric, colon, and pancreas, etc. (McManus et al., 1976;Yoshimura et al., 1994; Yamaguchi et al., 1989; Marcillac et al., 1992;Alfthan et al., 1992). Although the function of b-HCG production inthese tumors is still unknown, the atavistic expression of b-HCG bycancer cells and not by normal cells of non-gonadal origin suggests itmay be a potentially good marker in the detection of melanoma and breastcancer (Hoon et al., 1996; Sarantou et al., 1997).

Another exemplary example of a marker is glycosyltransferase b-1,4-N-acetylgalacto-saminyltransferase (GalNAc). GalNAc catalyzes thetransfer of N-acetylgalactosamine by b1(r) 4 linkage onto bothgangliosides GM3 and GD3 to generate GM2 and GD2, respectively (Nagataet al., 1992; Furukawa et al., 1993). It also catalyzes the transfer ofN-acetylgalactosamine to other carbohydrate molecules such as mucins.Gangliosides are glycosphingolipids containing sialic acids which playan important role in cell differentiation, adhesion and malignanttransformation. In melanoma, gangliosides GM2 and GD2 expression, areoften enhanced to very high levels and associate with tumor progressionincluding metastatic tumors (Hoon et al., 1989; Ando et al., 1987;Carubia et al., 1984; Tsuchida et al., 1987a), although gangliosides arealso expressed in melanoma, renal, lung, breast carcinoma cancer cells.The gangliosides GM2 and GD2 are immunogenic in humans and can be usedas a target for specific immunotherapy such as human monoclonalantibodies or cancer vaccines (Tsuchida et al., 1987b; Irie, 1985.)

Other markers contemplated by the present invention include cytolytic Tlymphocyte (CTL) targets. MAGE-3 is a marker identified in melanomacells and breast carcinoma. MAGE-3 is expressed in many melanomas aswell as other tumors and is a (CTL) target (Gaugler et al., 1994).MAGE-1, MAGE-2, MAGE-4, MAGE-6, MAGE-12, MAGE-Xp, and are other membersof the MAGE gene family. MAGE-1 gene sequence shows 73% identity withMAGE-3 and expresses an antigen also recognized by CTL (Gaugler et al.,1994). MART-1 is another potential CTL target (Robbins et al., 1994) andalso may be included in the present invention.

Preferred embodiments of the invention involve many differentcombinations of markers for the detection of cancer cells. Any markerthat is indicative of neoplasia in cells may be included in thisinvention. A preferred marker is an A908G estrogen receptor alphanucleic acid sequence and/or a K303R substitution in an estrogenreceptor alpha nucleic acid sequence.

XIX. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more chimeric polypeptides or chimericpolypeptides and at least one additional agent dissolved or dispersed ina pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one composition or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

In some embodiments, an effective amount of a compositoin of the presentinvention, such as an antagonist to an estrogen receptor alpha K303Rpolypeptide, is administered to a cell. In other embodiments, atherapeutically effective amount of a composition of the presentinvention is administered to an individual for the treatment of disease.The term “effective amount” as used herein is defined as the amount of acomposition of the present invention which is necessary to result in aphysiological change in the cell or tissue to which it is administered.The term “therapeutically effective amount” as used herein is defined asthe amount of a composition of the present invention that eliminates,decreases, delays, or minimizes adverse effects of a disease, such ascancer. A skilled artisan readily recognizes that in many cases thecomposition may not provide a cure but may only provide partial benefit.In some embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of a composition that provides a physiological change isconsidered an “effective amount” or a “therapeutically effectiveamount.”

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The composition may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived fiom inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments, the chimeric polypeptide is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

XX. Methods of Making Transgenic Mice

A particular embodiment of the present invention provides transgenicanimals that comprise constructs having the A908G mutation. In anotherembodiment, the transgenic animal comprises a polynucleotide encoding anestrogen receptor alpha amino acid sequence comprising K303R. Transgenicanimals expressing these mutations, recombinant cell lines derived fromsuch animals, and transgenic embryos may be useful in methods forscreening for and identifying agents that interact with the estrogenreceptor alpha, or affect breast tissue health.

In a general aspect, a transgenic animal is produced by the integrationof a given transgene into the genome in a manner that permits theexpression of the transgene. Methods for producing transgenic animalsare generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191;which is incorporated herein by reference), Brinster et al. 1985; whichis incorporated herein by reference in its entirety) and in“Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds.,Hogan, Beddington, Costantimi and Long, Cold Spring Harbor LaboratoryPress, 1994; which is incorporated herein by reference in its entirety).

Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

DNA clones for microinjection can be prepared by any means known in theart. For example, DNA clones for microinjection can be cleaved withenzymes appropriate for removing the bacterial plasmid sequences, andthe DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D™column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 mg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA.

Other methods for purification of DNA for microinjection are describedin Hogan et al. Manipulating the Mouse Embryo (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1986), in Palmiter et al. Nature300:611 (1982); in The Qiagenologist, Application Protocols, 3rdedition, published by Qiagen, Inc., Chatsworth, Calif.; and in Sambrooket al. Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989), all of which areincorporated by reference herein.

In an exemplary microinjection procedure, female mice six weeks of ageare induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby CO2 asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% C02,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

Randomly cycling adult female mice are paired with vasectomized males.FVB, C57BL/6 or Swiss mice or other comparable strains can be used forthis purpose. Recipient females are mated at the same time as donorfemales. At the time of embryo transfer, the recipient females areanesthetized with an intraperitoneal injection of 0.015 ml of 2.5%avertin per gram of body weight. The oviducts are exposed by a singlemidline dorsal incision. An incision is then made through the body walldirectly over the oviduct. The ovarian bursa is then torn withwatchmakers forceps. Embryos to be transferred are placed in DPBS(Dulbecco's phosphate buffered saline) and in the tip of a transferpipet (about 10 to 12 embryos). The pipet tip is inserted into theinfundibulum and the embryos transferred. After the transfer, theincision is closed by two sutures.

A skilled artisan is aware that transgenic mice are also commerciallyavailable, such as from Charles River Laboratories (Wilmington, Mass.).

EXAMPLES

The following examples are offered by way of example, and are notintended to limit the scope of the invention in any manner.

Example 1 Materials and Methods—Sample Preparation and NucleotideSequence Analysis

Histologic slides from archival, clinical specimens were screenedmicroscopically for evidence of hyperplasia. Microdissection ofspecimens was performed on 55 samples using serial sections fromformalin-fixed, paraffin-embedded tissue blocks as described (O'Connellet al., 1999). Briefly, alternative 3- and 10 um-thick sections were cutfrom the blocks and float mounted on glass slides. The 3-um-thick slideswere stained with hematoxylin-eosin and examined under the lightmicroscope to locate regions of normal and hyperplastic tissues; andthese areas outlined with a felt-tipped pen. The marked slide was thenused as a template to guide manual microdissection from thecorresponding regions of the unstained 10-um-thick sections. It waspossible to obtain distant normal tissue from 4 of the patients withhyperplasia. A skilled artisan recognizes that there are a variety ofmethods to isolate desired cells from nondesired cells other than bymanual manipulation or LCM. These include physical means of separatingout undesired cells from desired cells, such as by centrifugation basedon size, or centrifugation with magnetic beads attached to antibodiesspecific for desired and nondesired cell types.

DNA was liberated from the microdisseced specimens using a modificationof the procedure of O'Connell et al (1999). Genomic sequencing was thenperformed using PCR amplification of isolated DNA using ER primer 1(nucleotides 1093-1112 (5′ primer; 5′-CAAGCGCCAGAGAGATGATG-3′); SEQ IDNO:15) and ER primer 2 (nucleotides 1231-1250 (3′ primer);5′-ACAAGGCACTGACCATCTGG-3′; SEQ ID NO:16) of the ER gene (Greene et al.,1996). An aliquot of this amplification was then used to perform singlestranded PCR amplification using ER primer 3 (nucleotides 1221-1240 (3′primer); 5′-GACCATCTGGTCGGCCGTCA-3′; SEQ ID NO:17) of the ER gene. Afterprecipitation of the single stranded PCR amplification product,dideoxysequence analysis was performed using ER primer 4 (nucleotides1099-1119 (5′ primer); CAGAGAGAATGATGGGGAGGG-3′; SEQ ID NO:18). Inanother embodiment, an alternative ER primer is used in lieu of ERprimer 4, such as for nucleotides 1101-1130 (SEQ ID NO:35). Genomic DNAwas isolated from normal blood samples of 80 healthy women, and utilizedfor genomic sequence analysis as described above. RNA was also isolatedfrom four additional, frozen hyperplastic lesions, and utilized forRT/PCR amplification, cloning, and sequence analyses as described (Fuquaet al., 1991).

Example 2 Materials and Methods—Stable Transfection and Cell GrowthAnalyses

The WT ER expression construct was prepared in the pcDNAI vector asdescribed previously (Fuqua et al., 1995). Site directed mutagenesis ofthis construct was then utilized to generate the A908G transition andthe entire coding sequence of ER was verified by dideoxysequenceanalysis in this clone. The generation of stable transfectants wasperformed as described by Oesterreich et al. (1993) using cotransfectionwith the G418-selectable expression vector pSVneo at a ration of 25:1with the ER plasmids into MCF-7 breast cancer cells. To analyze forexpression of both WT or Var sequences, Western blot analyses wereperformed using the 6F11 antibody (DaKO). Two to three-fold elevatedlevels of total ER protein were detected in the two WT ER and the threeVar clones. In addition, RT/PCR amplification of cDNA from thetransfectants (Fuqua et al., 1991) followed by dideoxysequence analysisconfirmed that exogenous WT and Var RNA were expressed in the stabletransfectants. Furthermore, the relative levels of WT or Var sequenceswere determined by genomic sequence analysis as described above; the ERVar transfectants contained both WT nucleotide (A) and Var nucleotide(G) sequence in approximately equal ratios on the sequencing gels. Forcell growth studies, cells were plated at a density of 2×10⁴ in mediacontaining 10% charcoal-stripped, estrogen-free fetal calf serum andwere either left untreated or treated with the indicated increasingestradiol concentrations of 1×10⁻¹², 1×10⁻¹¹, or 1×10⁻⁹ M. The mediumwas replaced every 48 h and the cells were harvested and counted on days2, 4, 6, and 8, respectively.

Example 3 Statistical Methods

After taking logarithms to stabilize within group variances, asdetermined to be appropriate by Box-Cox analysis (Box and Cox, 1996),one-way analysis of variance was used to detect estrogen dose-relateddifferences in growth on Day 8 (i.e. 0 versus 10⁻¹² M versus 10⁻¹¹ Mversus 10⁻⁹ M), and to detect differences among estrogen doses (10⁻¹² Mversus 10⁻¹¹ M versus 10⁻⁹ M). The Student-Newman-Keuls multiple rangetest (α=0.02) was used to determine which doses were different from eachother. Analyses were preformed using SAS (V6.12, SAS Institute, Cary,N.C.).

Example 4 Materials and Methods—GST Pull-Down Assays

Bacterial expression vectors for GST-wt ER and GST-mutant ER wereconstructed by PCR amplification of the hinge and hormone bindingdomains of wild-type ER α and the A908G ER α using a sense primer(nucleotides 756-775 and an antisense primer (nucleotides 1788-1769)(Greene et al., 1996), and then cloning these products into theBamH1-EcoRI sites of pGEX-2kt GST gene fusion vector (Pharmacia). TheGST-pull down assays were performed as described (Ding et al., 1998)using recombinant SRC-2 (pSG5-human TIF-2) translated in vitro using theTNT coupled Reticulocyte Lysate System (Promega, Madison, Wis.), as wellas recombinant SRC-1 and SRC-3. The reactions were allowed to bind theglutathione-Sepharose 4B beads (Pharmacia) for 1.5 h in the presence ofincreasing amounts of estradiol at 4° C. Samples were subsequentlyanalyzed by SDS-polyacrylamide gel electrophoresis.

Example 5 Assay of Estrogen Receptor Alpha Sequence in Early BreastDisease

cDNA was prepared by reverse transcription of RNA from 4 typicalhyperplasias of the breast, to assay for an altered ER in early breastdisease, followed by polymerase chain reaction (PCR) amplification usingprimers specific for the entire coding domain of ERα (across nucleotides1182 to 1234). Cloning and sequencing of ER was performed as describedin Fuqua et al. (1991) except restriction sites were incorporated intothe primers to facilitate cloning into pGEM7zf (+) (Promega Corp.,Madison, Wis.). Wildtype ER sequence was identified in two of thesepremalignant lesions (FIG. 2). However, in the other two lesions an ER αvariant was identified with an A to G base pair transition at nucleotide908 (FIG. 2, top panel). This transition introduces a Lys to Argsubstitution at residue 303 within exon 4, at the border between thehinge domain D and the beginning of the hormone-binding domain E of ERα(FIG. 2, bottom diagram). Even though this substitution represents aconservative amino acid change, the size of the study was enlarged,since the data indicates that the amino-terminal region of the ERαhormone-binding domain is important in the generation of a completetranscriptional response in cells (Pierrat et al., 1994). Therefore,archival histological sections of 55 additional typical hyperplasiaswere microdissected, DNA was isolated, and direct genomic sequencing wasperformed using primers bordering ERα nucleotide 908. The same ER αalteration in 18/55 of these additional premalignant lesions wasidentified. Thus, the A908G ERα alteration was present in a total of20/59 (34%) of the hyperplasias examined.

DNA was prepared from normal breast epithelium adjacent to thehyperplastic lesion of those samples that contained the A908G ERαalteration. The ERα variant sequence was detected in the normal adjacentepithelium of some of these samples tested. Thus, the A908G ER αtransition is frequently present in premalignant lesions of the breast,and can occur in the adjacent normal-appearing breast epithelium.

Example 6

THE A908G ERα Mutation is a Somatic Mutation

To address whether the ER alteration might represent a somatic change inthe breast, rather than a germ-line alteration or a naturally-occurringpolymorphism within ERα, distant normal epithelium from 4 of the 20patients with the A908G ER alteration in their hyperplastic lesion wasmicrodissected. (Only 4 of the patients had sufficient normal distanttissue for analysis.) Manual microdissection on a light box under adissecting microscope was performed to microdissect archival,formalin-fixed, paraffin-embedded tissue blocks and was precise enoughto ensure at least 50% cellularity. DNA was liberated from themicrodissected specimens and direct genomic sequence analysis performed.Genomic sequencing of one patient's samples is shown in FIG. 3. VariantA908G ER α sequence was detected along with WT sequence in the normaladjacent DNA (N Adj.) and the typical hyperplasia (TH) DNA from thispatient, but the normal distant tissue (N Dis.) displayed only WT ER αsequence. All 4 of the patients with the variant ERα sequence in theirhyperplastic lesion exhibited WT sequence in their distant normaltissue. To further strengthen this observation, normal DNA was alsoexamined by direct genomic sequencing of 80 blood samples collected frompatients without breast disease. There was no detection of the ERαvariant sequence in any of these normal samples. Therefore, the A908GERa alteration is a somatic mutation appearing frequently in associationwith breast hyperplasia. Thus, just as LOH can occur in morphologicallynormal ductal epithelium adjacent to breast cancers (Deng et al., 1996),and may therefore demarcate a localized region predisposed to thedevelopment of breast cancer, in a specific embodiment a somaticmutation in ERα within a localized region of normal breast epitheliumdefines a region of increased risk if the mutation confers a selectiveadvantage to these cells.

Example 7 The A908G ERα Mutation Confers Selective Advantage to Cells

The proliferative response to hormones in breast cancer celltransfectants containing the mutation was tested to determine if this ERmutation confers a selective advantage. A CMV-driven mammalianexpression vector was prepared for WT ERα and utilized site-directedmutagenesis (Promega, Madison, Wis.) to generate the Lys303Argsubstitution. The mutant expression vector was stably introduced intothe ER-positive MCF-7 breast cancer cell line that normally expresses WTERα. This cell line was chosen because it was determined that WT ERα wasexpressed along with the mutant in the original 2/4 typical hyperplasticlesions which were examined. As a control, the expression vector wasalso stably transfected alone into MCF-7 cells. Transfected clones werethen cultivated in estrogen-depleted medium (−E₂) or medium supplementedwith increasing amounts of estradiol (10⁻¹² to 10⁻⁹M). Bothnon-transfected MCF-7 cells (FIG. 4, panel A) and vector-alonetransfected cells (panel B) exhibited typical estrogen dose responsegrowth curves. Minimal cell growth stimulation was seen with 10⁻¹²Mestradiol in these cells. Because it was possible that overexpression ofthe receptor alone might stimulate the growth of these cells, MCF-7cells were also transfected with the expression vector for WT ERα, buttheir estrogen dose response curves (FIG. 4, panels C and D) were notdifferent from the controls (Oesterreich et al., 1993). In contrast,three independent clones expressing the ERα mutation responded toextremely low levels of hormone (10⁻¹² M) (FIG. 4, panels E, F, and G)with nearly the same highly proliferative response seen at the highestconcentration of estradiol used (10⁻⁹ M).

Using analysis of variance (Box and Cox, 1996), it was determined thatthese were highly significant estrogen dose responses in the MCF-7,vector-alone transfected, and WT ERα-transfected cells (p=0.001), butthat there was little or no difference in response to differingconcentrations of estradiol in each of the three mutant ERα-transfectedclones (p=0.41, 0.015, and 0.09, respectively, for clones E, F, and G).The growth-stimulatory effects of low levels of hormone in cellsexpressing the ERα mutation were even more evident when doubling timeswere calculated from the growth curves. For example, the doubling timefor MCF-7 cells in 10⁻¹² or 10⁻⁹ M estradiol is 2.2 vs. 1.3 days,respectively. The doubling times for cells expressing the ERα mutant isthe same (1.3 days) at either 10⁻¹² or 10⁻⁹ M of hormone. Thus, theexpression of the ERα mutation confers a hypersensitivity to estrogenwith an ability to be maximally stimulated in response to physiologicallevels (10⁻¹² to 10⁻¹¹ M) of hormone. Thus, the A908G ERα mutation is again-of-function mutation that could have a significant biological rolein early breast disease.

In one embodiment, one mechanism by which the ERα mutation confershypersensitivity to low levels of hormone would be an increased bindingaffinity for estradiol. However, no differences in estradiol affinitywere detected between the WT ERα and the A908G ERα mutation usingsaturation binding Scatchard analyses, nor were there differences inaffinity for the antiestrogen tamoxifen.

In an alternative embodiment, one mechanism by which the ERα mutationconfers hypersensitivity to low levels of hormone might be alteredaffinity for ER co-regulators. It is now understood that many of thecell-type and tissue-specific effects of ERα are dependent on thecellular pool of co-regulatory factors that bind to and influence itstranscriptional activity (reviewed in Horowitz et al., 1997), many ofwhich act as signaling intermediates between the ER and the generaltranscriptional machinery, or directly have enzymatic activities such ashistone acetyltransferase activity. The A908G ERα mutation occurs in aregion implicated in binding to certain of these co-regulatory proteins,such as L7/SPA (Jackson et al., 1997) and the SRC-1 family ofco-activators (Onate et al., 1998). For example, efficient interactionof SRC-1 with the progesterone receptor hormone-binding domain requiresthe presence of hinge sequences (Onate et al., 1998). Thus, the abilityof WT and mutant ERα to interact with SRC-2 (TIF-2) (Voegel et al.,1996), a member of the SRC-1 family, was compared using in vitro GSTpull-down assays (Ding et al., 1998). GST-WT ERα and GST-ERα mutantfusion constructs containing the hinge and hormone binding domains wereprepared. Full-length SRC-1, SRC-2 and SRC-3 were synthesized in vitroin the presence of [³⁵S]methionine and then tested for specifichormone-dependent binding to the immobilized GST-ER fusion proteins(FIG. 5) by incubating with Sepharose beads containing immobilized GST,GST-WT ER, and GST-A908G mutant ER with or without estradiol. BoundSRC-1, SRC-2 and SRC-3 were eluted and observed by SDS-PAGE andautoradiography. Input SRC-1, SRC-2 and SRC-3 are shown (10%), as isnonspecific GST binding in the absence of estradiol. Increasing levelsof estradiol used were: 4×10^(−85×10) ⁻⁸, 6×10⁻⁸, 7×10⁻⁸, and 1×10⁻⁶M.Both receptors bound SRC-1, SRC-2 and SRC-3 in the presence (10⁻⁶ M),but not the absence of estradiol. However, the mutant required much lesshormone for efficient binding. Even at the lowest estradiolconcentration tested, 4×10⁻⁸M, the mutant ER efficiently bound SRC-2 andSRC-3, whereas WT ERα exhibited neglible binding at this concentration.The mutant ER also bound SRC-1 co-activator, although not to the sameextent as SRC-2 and SRC-3. This data indicates that the Lys303Argsubstitution enhances SRC-1, SRC-2 and SRC-3 binding by lowering theconcentration of hormone required to facilitate the formation of theco-activator:ER hydrophobic groove binding surface (Shiau et al., 1998)within the ER hinge/ligand binding domain. In another embodiment, anadditional mechanism includes this residue in the ER as a site foracetylation. An Arg substitution at this site could render it incapableof being acetylated, and/or the substitution could reduce the netnegative charge if surrounding Lys residues in the ER are indeedacetylated. Altered co-activator binding has also been reported for aTyr537Asn ERα mutation (Tremblay et al., 1998) that was identified in ametastatic bone lesion from a breast cancer patient (Zhang et al.,1997). Thus, it is important that both of these in vivo ERα mutationsdrastically affect the ability of the receptor to bind to co-regulatoryproteins.

A skilled artisan recognizes that there are alternative methods in theart to testing for acetylation in addition to immunodetection methods.

Example 8 Single Strand Conformation Polymorphism (SSCP) Analysis of ERMutation

A skilled artisan recognizes that there are multiple methods known inthe art to identify a mutation, including SSCP. Additional clinicalsamples were examined by manually microdissecting permanent sections of10 typical hyperplasias. Manual microdissection on a light box under adissecting microscope was performed to microdissect archival,formalin-fixed, paraffin-embedded tissue blocks and was precise enoughto ensure at least 50% cellularity. DNA was liberated from themicrodissected specimens as described (Fuqua et al., 1991) and SSCPanalysis performed (Orita et al., 1989) using primers spanning across ERnucleotide 908 (FIG. 6). SSCP was performed as previously described(Elledge et al., 1993) except ER primers were used for PCR amplification(nucleotides 1093-1112 (5′ primer; SEQ ID NO:15) and 1231-1250 (3′primer; SEQ ID NO:16) of the ER gene (Greene et al., 1986). The gelswere electrophoresed in 0.5×TBE at room temperature for 14 h. To bescored as having an alteration, a DNA sample had to produce an abnormalSSCP pattern using separate DNA aliquots and amplified on different dayswith negative controls.

Five of the hyperplasias (samples 2, 4, 5, 7, and 8) displayed bandmobilities which were identical to those of the complementary strands ofthe PCR fragment from the WT ER control DNA. However, in five of thehyperplasias (samples 1, 3, 6, 9, and 10) four bands could be detected.These results indicated that the DNA from these later five hyperplasiashad two different ER alleles, one WT and the other migrating identicalwith the mutant (Mut) ER allele. Further proof that these fastermigrating bands contained the A908G transition was obtained by cuttingthe region corresponding to the Mut band from the dried gel, cloning thefragment, and dideoxysequence analyzing to confirm.

Example 9 Oligonucleotide Mismatch Mutation Detection

A sensitive oligonucleotide mismatch hybridization method (Moul et al.,1992) was used to detect the ER alleles in a cancer patient. Inaddition, laser capture microdissection was utilized to more preciselyenrich for the specific lesions present concomitantly in this patient.

A nested PCR amplification procedure was used to amplify the lasercapture microdissected material (Bonner, 1997) where the outside primerscorrespond to those used in the SSCP analysis described above in a 30 μlreaction volume, and then 1.5 μl of this was then reamplified with ERprimer sequences corresponding to nucleotides 1101-1130 (5′) and1220-1239 (3′) of the ER gene (Greene et al., 1986). The samples werethen denatured in 0.4 M NaOH, 25 mM EDTA at 95° C., then neutralizedwith 1 M Tris-HCl pH 7.4 before slotting on the nylon membranes.Oligonucleotide probes corresponding to the WT (SEQ ID NO:33;5′-GCTCTAAGAAGAACAGCCTG-3′) or Mutant (SEQ ID NO:34;5′-GCTCTAAGAGGAACAGCCTG-3′) (corresponding to nucleotides 1191 to 1210of the ER gene (Greene et al., 1986)) were end-labeled with T4 kinase.The membrane was prehybridized in 5×SSPE, 0.5% SDS, 5×Denhardt's andwashed at 60° C. 2×SSPE, 0.1% SDS followed by a wash at 68° C. in5×SSPE. 0.1% SDS. Control WT or Mut plasmid DNAs were also amplified,slotted, and hybridized as positive controls for hybridization; sampleswithout added DNA were included as negative controls duringamplification.

The variant sequence was detected in the normal adjacent breastepithelium (AB), the hyperplastic lesion (H), and one ductal carcinomain situ (DCIS) lesion using an oligonucleotide probe specific for thevariant, but not in normal skin (NS), normal distant breast epithelium(DB), or another independent DCIS lesion in this patient (FIG. 7, rightpanel). Both WT (FIG. 7, left panel) and mutant ER alleles were presentin this patient.

Example 10 Incidence of the A908G Mutation in Invasive Breast Cancers

In a specific embodiment of the present invention, breast cancer samplesfrom invasive breast tumors are assayed by standard methods, such asthose described herein, for the A908G mutation in estrogen receptoralpha nucleic acid sequence. A skilled artisan recognizes that there arepresently two types of invasive breast cancer: Node-negative andNode-positive. In approximately half of women with invasive breastcancer, the lymph nodes are invaded (Node-positive), and there are alsomicrometastases elsewhere within the body. In approximately half ofwomen with invasive breast cancer, the cancer has not spread to thelymph nodes.

Ca. from Node-positive Ca. from Node-negative women women Wild-type 16 4Mutant 10 23 (p = 0.00062 Fisher's Exact Test, two sided)

Therefore, the frequency of the mutation in invasive breasttumors=33/53=62%. Thus, the A908G mutation is identified in bothNode-negative and Node-positive invasive breast cancers.

Example 11 Screening for Antagonists and Agonists of ERα K303RPolypeptide

In some embodiments of the present invention, candidates for drugs arescreened which are useful for treatment of a breast cancer related tothe A908G mutation in ERα polynucleotide and/or the ERα K303Rpolypeptide which it encodes. In specific embodiments, antagonists oragonists are screened for which affect the activity of the ERα K303Rpolypeptide.

A skilled artisan recognizes that a variety of methods known in the artare available to screen for antagonists or agonists of ERα K303Rpolypeptide. For example, transfection assays are utilized (such asdescribed in Barkhem et al. (1997); Cowley et al. (1997); and Sun et al.(1999), all of which are incorporated by reference herein in theirentirety) wherein a cell is transiently or stably transfected with anexpression vector comprising the ER form to be tested against, areporter expression construct operably linked to at least one estrogenresponse element, such as 5′-AGGTCA-3′ (SEQ ID NO:36); 5′-TGACCT-3′ (SEQID NO:37); 5′-GGTCAnnnTGACC-3′ (SEQ ID NO:38); 5′-AATCAnnnTGACT-3′ (SEQID NO:39); 5′-GGTCA-3′ (SEQ ID NO:40); 5′-TGGTC-3′ (SEQ ID NO:41);5′-TGACC-3′ (SEQ ID NO:42); 5′-ATTCGATCAGGGCGGGGCGAGC-3′ (from SP1; SEQID NO:43); 5′-GGGCA(N)₁₆GGCGGG-3′ (c-myc; SEQ ID NO:44);5′-GGTCA(N)₂₁GGCGG-3′ (ckb; SEQ ID NO:45); 5′-GGGCCGGG(N)₁₀GGTCA-3′(cathepsin D; SEQ ID NO:46); 5′-GGGCA-3′ (hsp27; SEQ ID NO:47);5′-GGTAA-3′ (cathepsin D; SEQ ID NO:48); 5′-GGTCA(N)3TGCCC-3′(uteroglobin; SEQ ID NO:49); 5′-GGGGCGTGG-3′ (c-fos; SEQ ID NO:22);5′-CCGCCCC-3′ (e2f; SEQ ID NO:26); 5′-TGA(C/G)TCA-3′ (AP1; SEQ ID NO:8).A compound to be tested is administered to the cell, and the expressionlevel of the reporter expression construct is assayed in the presence ofthe test compound and compared to expression levels in its absence. Atest compound which downregulates expression of the reporterpolynucleotide is considered an antagonist, and a test compound whichupregulates expression of the reporter polynucleotide is considered anagonist.

In alternative embodiments for drug/antagonist/agonist screening, a twohybrid assay is performed, such as is described in Slentz-Kesler et al.(2000), incorporated by reference herein in its entirety. In a specificembodiment, a polynucleotide encoding the ERα K303R polypeptide as afusion polypeptide with a DNA binding domain is transformed into a yeastor mammalian cell. The population of corresponding yeast or mammaliancells further comprise a library of expression vectors producingchimeric polypeptides comprising a DNA activation and a librarycandidate. Interaction of the ERα K303R polypeptide with a particularlibrary candidate is visualized by assaying expression of a reportersequence expression influenced by the interaction of the correspondingDNA activation and binding domains. A skilled artisan recognizes thatmultiple DNA activation and binding domains are available, includingGAL4 or LexA. Also, controls are performed to eliminate any falsepositives.

In another embodiment to identify and design drugs for ERα K303Rpolypeptide-associated breast cancer, particularly antagonists andagonists, a phage peptide display assay is employed, such as isdescribed in Sparks et al. in Phage Display of Peptides and Proteins, ALaboratory Manual (Academic, San Diego), incorporated by referenceherein. In this embodiment, an affinity-tagged labeled ERα K303Rpolypeptide is exposed to a nitrocellulose membrane comprisingbacteriophage plaques each of which comprise a peptide. Binding of theERα K303R polypeptide to the peptide is assayed, and the resultantpeptides are identified. In some embodiments, the affinity selection ofthe phage-displayed peptide libraries is conducted on the ERα K303Rpolypeptide in different conditions, such as in an apo form,ligand-bound form, and so forth. The resultant peptides are analyzed,allowing rational drug design to ensue based on the analysis.

In an additional embodiment, other methods are known to evaluate theeffects of an antagonist vs. an agonist of a receptor-binding substanceon a selected type of cells containing an endogenouse intra-cellularhormone receptor, such as is described in U.S. Pat. No. 5,578,445,incorporated by reference herein. Therein, an in vitro method isdisclosed wherein a test substance and a reference substance, known tobe either an antagonist or an agonist of the receptor, is incubated withcells, and the magnitude of the selected cellular response resultingfrom the hormone/receptor interaction is analyzed.

In another embodiment, drug candidates/antagonists/agonists for ERαK303R polypeptide are analyzed by mass spectrometry (Witkowska et al.,1997) or by X-ray crystallography (Shiau et al., 1998), both of whichare incorporated by reference herein in their entirety. A skilledartisan recognizes that the National Center for BiotechnologyInformation provides a structural database(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed) containingmany protein structures, including estrogen receptor. Analyses of theERα K303R polypeptide by these methods provide significant structuraldetail so that, for example, an antagonist to fit a particularstructural domain can be designed. In one embodiment, X-raycrystallography is performed on the ERα K303R polypeptide bound to aspecific ligand, such as estradiol, tamoxifen, raloxifene, droloxigene,GW 5638, idoxifene, CP336156, or LY353381. Such an analysis facilitatesdesign of a drug which will antagonize the activity of the ERα K303Rpolypeptide, such as for the treatment of breast cancer. For massspectrometry methods to facilitate drug screen/antagonist/agonistanalysis, methods may be employed which are similar to Witkowska et al.(1997), wherein structural comparisons were made between twostructurally similar compounds. In a particular embodiment, the massspectrometry analysis provides information on binding sites forcofactors.

In one embodiment of the present invention, there is a method ofdesigning an agent which affects the activity of an estrogen receptoralpha K303R polypeptide, comprising determining the crystal structure ofa purified estrogen receptor alpha K303R polypeptide; and analyzing amodel of the crystal structure, wherein the agent is designed based onthe analysis.

In another embodiment of the present invention there is a method ofdesigning an agent which affects the activity of an estrogen receptoralpha K303R polypeptide, comprising determining the crystal structure ofa purified estrogen receptor alpha K303R polypeptide in the presence ofa compound which interacts with the estrogen receptor alpha K303Rpolypeptide; and analyzing a model of the crystal structure, wherein theagent is designed based on the analysis. In a specific embodiment, theanalyzing step comprises computer modeling. In another specificembodiment, the crystal structure is determined in the presence of anestrogen receptor ligand.

In an additional embodiment of the present invention, there is a methodof designing an agent which affects the activity of an estrogen receptoralpha K303R polypeptide, comprising analyzing the structure of thepolypeptide by mass spectrometry, wherein the structure of thepolypeptide suggests the design of the activity-affecting agent. In aspecific embodiment, the activity-affecting agent is an antagonist. Inanother specific embodiment, the activity-affecting agent is an agonist.

Example 12 Significance of the Present Invention

In summary, it is shown that a large proportion of premalignant breasthyperplasias express an altered ERα that is hypersensitive to theeffects of estrogen. Furthermore, the alteration results from a somaticmutation in the breast with this mutation affecting the ability of thereceptor to bind to the SRC-1, SRC-2, and SRC-3 co-activators. There isan increasing body of evidence, both epidemiological (Dupont and Page,1985) and molecular (O'Connell et al., 1998), suggesting that thesepremalignant lesions are both risk factors and direct precursors ofinvasive breast cancer. However, hyperplasias are relatively common inthe breast, and only a small fraction of them will progress to cancer.Prior to the methods and compositions of the present invention, those inthe art have been unable to differentiate which of these lesions aregenetically stable, or the biological differences driving some of themto progress. An ERα mutation that confers a proliferative advantage,such as hypersensitivity to hormone, in a specific embodiment provides afavorable cellular environment accelerating the accumulation ofadditional genetic events important for tumor progression.

Premalignant breast lesions are microscopic masses with a positivegrowth imbalance, and the hypersensitive ERα mutation is likely animportant factor contributing to this imbalance. Hormone levels normallyfluctuate during the menstrual cycle in premenopausal women, and levelsare considerably lower in postmenopausal women. In one embodiment, an ERmutation hypersensitive to estradiol provides a continuous mitogenicstimulus to the breast epithelium even during phases of low circulatinghormone, especially in postmenopausal women, thus elevating their riskfor breast cancer. Thus, in a preferred embodiment, there is acorrelation between risk for breast cancer and expression of this ERαmutation, which will allow genetic analysis for the mutation inpremalignant lesions to be crucial to identify patients who wouldbenefit from preventive measures.

Example 13 Ductal Hyperplasias in K303R Transgenic Mice

Transgenic mice expressing the K303R mutation were generated by standardmeans in the art. The mice at the time of filing of the nonprovisionalapplication have matured to 18 months, and they have developed ductalhyperplasias (FIG. 8, panels A through D). Nontransgenic mainmary glandsare shown in panels 8E and 8F. The H&E-stained histological sectionsshown in panels 8A and 8B clearly demonstrate the development of ductalhyperplasias in the transgenic mice with luminal epithelial cellsbeginning to stratify in the ductal lumen in the mammary glands. Panel8B shows a duct whose lumen is completely filled with epithelial cells.In a specific embodiment of the present invention, the hypersensitive ERmutation provides a proliferative advantage, especially by providing acontinuous mitogenic stimulus to the epithelium even in an environmentof low circulating hormones, such as these virgin mice.

Ductal hyperplasias are composed of both an increase in the number ofepithelial cell layers within the duct (shown in panel 8C), as well asan increase in the number of small ducts within a given area (shown inpanel 8D). These increases in the transgenic animals are more clearlyobserved when one compares the histological sections from nontransgenicmammary glands (shown in panels 8E and 8F).

FIG. 9 shows that the K303R transgenic animals have increasedproliferation as compared to nontransgenic animals in the ductalepithelium. Proliferation was measured by immunohistochemistry with anantibody to phosphorylated histone H1b, a surrogate marker of S-phase.

Thus, the data in FIGS. 8 and 9 show that expression of the K303Rmutation, which was originally identified in human breast hyperplasticlesions, is indeed an important factor contributing to abnormal ductalgrowth and the development of proliferating ductal hyperplasias.

REFERENCES

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referenceherein.

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One skilled in the art readily appreciates that the patent invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Mutations, kits,sequences, methods, procedures and techniques described herein arepresently representative of the preferred embodiments and are intendedto be exemplary and are not intended as limitations of the scope.Changes therein and other uses will occur to those skilled in the artwhich are encompassed within the spirit of the invention or defined bythe scope of the pending claims.

1. An isolated nucleic acid molecule comprising SEQ ID NO:34.
 2. Thenucleic acid molecule of claim 1, wherein said molecule is between 20and 100 nucleotides.
 3. The nucleic acid molecule of claim 1, whereinsaid molecule is from 20 to 30 nucleotides in length.
 4. The nucleicacid molecule of claim 1, wherein said molecule is up to 1-2 kilobasesin length.
 5. The nucleic acid molecule of claim 1 wherein said nucleicacid sequence is a DNA.
 6. The nucleic acid molecule of claim 1 whereinsaid nucleic acid sequence is a RNA.
 7. The nucleic acid molecule ofclaim 1, wherein said molecule is about 21 nucleotides in length.
 8. Thenucleic acid molecule of claim 1, wherein said molecule is about 22nucleotides in length.
 9. The nucleic acid molecule of claim 1, whereinsaid molecule is about 23 nucleotides in length.
 10. An isolated nucleicacid molecule that has 90% or more identity to the full length of SEQ IDNO:6 and comprises SEQ ID NO:34, or the complement thereof.